Lablogatory

Microbiology Case Study: Young Female with Facial Lesions

Case Presentation

A young female presented to the dermatology clinic with a 6-month history of two worsening painless lesions on her nose and left cheek but was otherwise well. The lesions appeared while the patient was living in Ethiopia. On physical examination, two irregular, bumpy, yellow-brown lesions with surrounding erythematous papules (Figure 1A) were documented. Erythema and hyperpigmented patches surrounding the lesions were also noted. Both lesions were non-pruritic, and no other cutaneous or mucosal lesions were observed. A punch biopsy was obtained, and the lesion was sent for culture and histopathological review.

Laboratory Workup

Histopathological examination of the biopsy specimen revealed granulomatous inflammation with numerous intracellular and extracellular Leishmania sp. amastigotes (Figure 2A). The amastigotes measured 2–4 µm in diameter and were oval to round with a defined nucleus and kinetoplast (Figure 2B). A diagnosis of cutaneous leishmaniasis (CL) was made, and the patient was referred to the infectious disease (ID) clinic for further evaluation and management. The patient was prescribed miltefosine for 28 days with planned follow up with ID, dermatology and ear nose and throat (ENT) groups. At a subsequent follow up visit two months later, her lesions have visibly improved, and she continues to be followed outpatient (Figure 1B).

Figure 1. Clinical presentation of a young woman with Leishmaniasis. Photographs of the patient’s left cheek at initial presentation (A) and following 28 days of miltefosine treatment (B).

Figure 2. Histopathology of *Leishmania* in the patient’s biopsy from the left cheek (H&E). A) A dense dermal infiltrate composed of histiocytes and lymphocytes with numerous forms consistent with amastigotes of *Leishmania* (orange boxes) within histiocytes (400x magnification). B) High magnification of amastigote-containing histiocytes with observable kinetoplasts (orange arrows) (1000x magnification, oil immersion).

Discussion

Leishmaniasis is a vector-borne disease transmitted by sandflies. The disease is caused by obligate intracellular protozoan parasites of the genus Leishmania. Human infection is caused by 21 of 30 known species that infect mammals in the Eastern and Western hemispheres. Examples of common species causing disease in the Eastern hemisphere include the L. donovani complex, L. tropica, L. major, and L. aethiopica whereasthe most common species found in the Western hemisphere include L. mexicana complex, L. braziliensis, and the subgenus Viannia.¹ The different species are morphologically indistinguishable and can only be differentiated by isoenzyme analysis, molecular methods, or monoclonal antibodies.²

Leishmaniasis presents as a diverse range of diseases depending on the species associated with the infection. Visceral Leishmaniasis (VL) is associated with L. donovani complex in the Eastern hemisphere and L. chagasi in the Western, can be life threatening, and is characterized by fever, weight loss, hepatosplenomegaly, and pancytopenia.¹ More than 90 percent of the world’s cases of VL are in India, Bangladesh, Nepal, Sudan, and Brazil.² Worldwide, it has an estimated annual incidence of 0.7–1.0 million cases.³ The disease primarily affects the skin and can result in disfiguring lesions.

CL is the most common manifestation observed worldwide. L. major, L. tropica and L. aethiopica are common causes of CL in the East and L. braziliensis and L. mexicana are common causes of CL in the West.¹ The clinical presentation of cutaneous leishmaniasis can vary depending on the Leishmania species involved, the immune status of the host, and the local immune response at the site of infection. The incubation period of cutaneous leishmaniasis can range from several days to months, with disease presenting as solitary or multifocal lesions. Affected areas may be ulcerated or nodular.⁴ This patient’s presentation was more nodular in appearance. Additionally, L. braziliensis can cause mucocutaneous leishmaniasis frequently months to years after spontaneously healed CL, which can erode the nasal septum, palate, or other mucosal structures.²

Serological tests can detect the presence of antibodies against Leishmania but cannot be used as a surrogate for treatment success because antibodies can continue to circulate following successful treatment.⁵ Histopathological examination of a skin biopsy is the gold standard for the diagnosis of cutaneous leishmaniasis. The presence of intracellular and extracellular amastigotes in a granulomatous inflammatory infiltrate is highly suggestive of the disease.⁶ Treatment of leishmaniasis depends on the presentation and disease severity. Topical therapy, such as paromomycin ointment, can be used for CL. Systemic therapy (such as what this patient received) is indicated in the setting of immunosuppression, or if the lesions are large, multifocal, affect the joints, hands, or feet. Various antiparasitic agents are available for the treatment of leishmaniasis, including pentavalent antimonials. amphotericin B, miltefosine, and paromomycin.⁶ In some cases, surgical intervention may be required to remove larger ulcers or nodules.

References

1. Garcia, L. Diagnostic Medical Parasitology, 6^th edition. “Leishmaniasis”. ASM Press. 2016. Chapter 27, p778-793.

2. CDC – DPDx – Leishmaniasis. Published January 18, 2019. Accessed February 20, 2023. https://www.cdc.gov/dpdx/leishmaniasis/index.html

3. Burza S, Croft SL, Boelaert M. Leishmaniasis. Lancet. 2018;392(10151):951-970. doi:10.1016/S0140-6736(18)31204-2

4. Reithinger R, Dujardin JC, Louzir H, Pirmez C, Alexander B, Brooker S. Cutaneous leishmaniasis. Lancet Infect Dis. 2007;7(9):581-596. doi:10.1016/S1473-3099(07)70209-8

5. Aronson NE, Joya CA. Cutaneous Leishmaniasis: Updates in Diagnosis and Management. Infect Dis Clin North Am. 2019;33(1):101-117. doi:10.1016/j.idc.2018.10.004

6. Handler MZ, Patel PA, Kapila R, Al-Qubati Y, Schwartz RA. Cutaneous and mucocutaneous leishmaniasis: Differential diagnosis, diagnosis, histopathology, and management. J Am Acad Dermatol. 2015;73(6):911-926; 927-928. doi:10.1016/j.jaad.2014.09.014

–L. Jonathan He is a fourth year AP/CP resident at UT Southwestern Medical Center in Dallas, Texas.

-Dominick Cavuoti, DO is a professor in the Department of Pathology who practices Medical Microbiology, Infectious Diseases Pathology and Cytology.

-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.

A Traveler from Southern America with Nightly Fevers and Dysuria

Case Presentation

A 26 year old male presented to the emergency department (ED) with 1 week of malaise, high nightly fevers, abdominal pain, dark urine, and dysuria. He had no past medical history and was not taking any medications. He had been living in shelters since arriving in the US approximately 1 month ago after several months traveling through South and Central America from Venezuela. On review of systems, he also reported 3-4 days of constipation and a single episode of non-bilious, non-bloody vomit 2 days ago, accompanied by nausea. He denied mosquito bites, bloody or dark stools. On physical exam, patient appeared thin, mildly jaundiced, and had LUQ/LLQ moderate tenderness to palpation without guarding and splenomegaly. Given his travel history and presenting symptoms, the infectious disease service was consulted, and blood was drawn in the ED for further analysis. CBC and CMP were significant for normal WBC (10.21 x 10³/mcL), normocytic anemia (hemoglobin 11.3 gm/dL, MCV 80.4 femtoliters), thrombocytopenia (48 x 10³/mcL), hyperbilirubinemia (total bilirubin 2.4 mg/dL, indirect bilirubin 1.7 mg/dL), and slightly elevated AST (46 units/L). Thin blood smear showed relatively enlarged erythrocytes infected with multiple ring forms and trophozoites with ameboid cytoplasm, consistent with Plasmodium vivax. Parasitemia was calculated to be 0.4%. The patient was diagnosed with uncomplicated malaria and started on artemether-lumefantrine for 3 days. He was found to a severe deficiency (41 units/10¹² RBC) of glucose-6-phosphate dehydrogenase (G6PD) and therefore, at high risk of hemolysis if administered primaquine. Given his complex social situation, the patient decided to monitor himself for symptoms of relapse and defer starting primaquine for treatment of the liver stages of P. vivax. He was discharged to a local shelter with plans to follow-up closely with the outpatient infectious disease department, with strict precautions to return to the ED urgently for recurrent symptoms.

Figure 1. Thick (left) and thin (right) peripheral blood smears from patient reveal large infected red blood cells and “amoeboid” forms suggestive of *P. vivax*. Photo taken by Dr. Zoon Tariq, PGY3, Department of Pathology at The George Washington University School of Medicine and Health Sciences.

Discussion

Malaria should be suspected when a patient presents with a febrile illness and a travel history within a malaria-endemic region. Diagnosis of P vivax can be made through microscopic examination of blood smears, immunochromatographic rapid diagnostic tests (RDTs) and nucleic acid detection through amplification techniques.¹ Examination of a thick blood smear allows efficient screening for malaria parasites, while a thin blood smear allows for species identification since parasite morphology is more clearly visualized.² Upon examination of thin blood smears, infections by P. vivax and P. ovale may appear indistinguishable as both species infect immature, enlarged erythrocytes (1.25-2x normal), can be visualized at any stage in peripheral blood (ring, trophozoite, schizont, and gametocyte) and because Schuffner’s dots are a common morphologic feature during most stages. Defining characteristics of P. vivax include the presence of a large, ameboid trophozoite cytoplasm, and fine Schuffner’s dots and schizonts with >12 merozoites. Of note, preparation with Giemsa stain over Wright stain is preferred for demonstration of Schuffner’s dots.² Immunochromatographic RDTs detect parasite-specific antigens (e.g., Plasmodium lactate dehydrogenase, Plasmodium specific aldolase) in a finger-prick blood sample. These tests are commercially available and relatively simple to perform and interpret, making them a useful tool for resource-limited regions.¹ Nucleic acid amplification-based tools (e.g., PCR, loop-mediated isothermal amplification) are not routinely used for clinical management of malaria but do have diagnostic advantages over light microscopy and RDTs.³ PCRs are highly sensitive, can detect mixed infections even at low parasite densities, and are useful for epidemiological studies such as drug resistance identification.¹

Malaria is a potentially fatal, but preventable and treatable, disease caused by infection of erythrocytes with protozoan parasites of the Plasmodium genus. Parasites are transmitted by the bites of infected female Anopheles mosquitos. Five species of Plasmodium (P. falciparum, P. vivax, P. ovale, P. malariae, P. knowlesi) infect humans. While each species of Plasmodium has unique characteristics, all species follow a similar life cycle. Erythrocyte lysis and release of merozoites cause release of pyrogens and production of inflammatory cytokines such as IL-1 and TNF-alpha, resulting in the symptomatic presentation of Plasmodium sp. infection, termed malaria. An important distinction between P. vivax and P. ovale and other Plasmodium species is that P. vivax/ovale may remain dormant in the liver (“hypnozoites”) and may resume intrahepatic replication, causing relapse of malaria weeks to years later.⁴ According to the WHO Malaria Report 2021, approximately half the world’s population lives in areas at risk of malaria infection. P. vivax is found predominately in Asia, Latin America, and some parts of Africa. It is the most common infective species in Latin America, accounting for an estimated 71.5% of cases in 2021.

The typical incubation time between transmission of parasites and onset of disease is approximately 14 days. P. vivax parasitesprimarily infect immature erythrocytes, representing 1-2% of the cell population. Synchronous replication and rupture of infected erythrocytes leads to the hallmark clinical presentation of cyclical severe fever and chills. Classically, P. vivax and P. ovale present with “tertian” malarial paroxysms, with fever and chills occurring every 48 hours. Patients may also complain of fatigue, malaise, headache, diaphoresis, abdominal pain, myalgias, dark urine, nausea, and vomiting. Additional clinical features and complications include anemia, jaundice, splenomegaly, and hepatomegaly. Severe infection presents with hemodynamic instability, pulmonary edema, coagulopathy, organ failure, neurological dysfunction, and potentially death.^{4, 5}

Chloroquine or artemisinin-based combination therapy (ACT) are both effective treatments against uncomplicated, non-falciparum malaria (P. vivax, P. ovale, P. malariae, P. knowlesi).In a large systematic review, ACTs were found to be at least equivalent to chloroquine when treating the blood stage of P. vivax infection.⁶ ACTs are the drug of choice for non-falciparuminfections in countries where chloroquine resistance has developed, notably New Guinea and Indonesia.⁷To prevent relapse caused by hypnozoites of P. vivax/ovale, initial treatment is followed by administration of primaquine. Before treatment initiation with primaquine, quantitative testing for glucose-6-phosphate dehydrogenase (G6PD) deficiency should be completed since this drug may induce hemolysis in those who are deficient. A modified dosing schedule under close medical supervision is used for those who are G6PD deficient. Treatment of severe malaria is with at least 24 hours of intramuscular or intravenous artesunate, with an option to transition to an ACT regimen once oral therapy can be tolerated.¹

Malaria caused by P. falciparum is typically more severe than malaria caused by P. vivax since P. falciparum infects erythrocytes of all ages, causing extensive hemolysis and related complications.^{4, 5} P. vivax malaria may also cause severe malaria and also relapses, emphasizing the importance of radical cure of hypnozoites with primaquine.⁸ Expedient and appropriate treatment leads to resolution of fever and parasitemia within days, with successful treatment confirmed by undetectable parasitemia on blood smear. Recurrence of a febrile illness should prompt re-evaluation and may suggest recrudescence or relapse due to failed therapy, or reinfection.

References

1. World Health Organization. (‎2023)‎. WHO guidelines for malaria, 14 March 2023. World Health Organization. https://apps.who.int/iris/handle/10665/366432.

5. Despommier DD, Griffin DO, Gwadz RW, Hotez PJ, Knirsch CA. Chapter 9: The Malarias. In: Parasitic Diseases. 7th ed. Parasites Without Borders, Inc.; 2019:93-122.

4. Ryan KJ. Chapter 51: Apicomplexa and Microsporidia. In: Sherris & Ryan’s Medical Microbiology. 8^th ed. McGraw Hill; 2022. Accessed June 20, 2023. https://accessmedicine-mhmedical-com.proxygw.wrlc.org/content.aspx?bookid=3107&sectionid=260929904

2. DPDx – Laboratory Identification of Parasites of Public Health Concern: Malaria. Centers for Disease Control and Prevention. Updated: October 6, 2020. Accessed: June 20, 2023. https://www.cdc.gov/dpdx/malaria/index.html

3. World Health Organization. Malaria Policy Advisory Group: Meeting Report of the Evidence Review Group on Malaria Diagnosis in Low Transmission Settings. Geneva, Switzerland: WHO Headquarters, 2013. Accessed June 20, 2023. https://www.who.int/publications/m/item/meeting-report-of-the-evidence-review-group-on-malaria-diagnosis-in-low-transmission-settings

6. Gogtay N, Kannan S, Thatte UM, Olliaro PL, Sinclair D. Artemisinin-based combination therapy for treating uncomplicated Plasmodium vivax malaria. Cochrane Database Syst Rev. 2013;2013(10):CD008492. Published 2013 Oct 25. doi:10.1002/14651858.CD008492.pub3

7. Price RN, von Seidlein L, Valecha N, Nosten F, Baird JK, White NJ. Global extent of chloroquine-resistant Plasmodium vivax: a systematic review and meta-analysis. Lancet Infect Dis. 2014;14(10):982-991. doi:10.1016/S1473-3099(14)70855-2

8. Dini S, Douglas NM, Poespoprodjo JR, et al. The risk of morbidity and mortality following recurrent malaria in Papua, Indonesia: a retrospective cohort study. BMC Med. 2020;18(1):28. Published 2020 Feb 20. doi:10.1186/s12916-020-1497-0

-Cleo Whiting is a fourth-year medical student at George Washington School of Medicine and Health Sciences. Her research interests include infectious disease, autoimmune disease, and dermatopathology.

-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.

A Diamond in the Mucin

A 35 year old male and current every day smoker transferred his care to our genitourinary (GU) clinic after undergoing a partial nephrectomy for clear cell renal cell carcinoma. A PET scan was performed, and a 5 millimeter FDG-avid right upper lobe (RUL) lung nodule was identified. While the mediastinal and hilar lymph nodes were mildly enlarged on the chest CT, they did not demonstrate FDG-avidity on the PET scan. The patient was referred to pulmonary for a workup of his subcentimeter lung nodule. Given the patient’s age and clinical history, the care team and the patient decided to pursue a lung biopsy via robotic-assisted bronchoscopy and subsequent lymph node sampling via endobronchial ultrasound (EBUS).

After the timeout, the patient was intubated, and the pulmonologist performed an airway inspection. Upon entering the right upper lobe, the pulmonologist noticed blood streaking along the bronchial wall. A right upper lobe bronchoalveolar lavage (BAL) was collected and sent for culture and cytology. Following the trachea and lung survey, the respiratory team began the robotic-assisted bronchoscopy. After confirming the correct location with both radial EBUS and fluoroscopy, multiple fine needle aspirates (FNAs) were collected from the right upper lobe lung nodule. Only benign bronchial cells were identified on rapid onsite evaluation (ROSE). A right upper lobe bronchial brushing was attempted and sent directly to cytology. Forceps were used to obtain transbronchial biopsies from the radiologically-suspicious area. The team then switched over to linear EBUS for the lymph node sampling portion of the procedure. Lymph nodes were sampled at the following stations: 11L, Level 7, and 4R. On ROSE, only lymphocytes and anthracotic pigment were identified for all three lymph nodes.

Upon e, the cytologist brought the specimens back to the laboratory for accessioning and processing. The following morning, the same cytologist screened all cytology specimens and the respective cell blocks associated with the case. The lymph nodes were signed out as negative for malignancy, with lymphocytes and anthracotic pigment present. The RUL FNA was inadequate for diagnosis as the material consisted mainly of bronchial cells and alveolar macrophages. The FNA findings were correlated with the transbronchial biopsy, which was signed out as benign pulmonary parenchyma with hemorrhage, fibrin clot, and respiratory bronchiolitis. Similarly, the bronchial brushing was negative as only benign bronchial cells were identified. In screening the BAL, there was an overabundance of hemosiderin-laden alveolar macrophages on the smear, cytospin, and SurePath liquid-based preparations (Image 1). When screening the cell block slides, abundant macrophages were also identified (Image 2).

Images 1-2. Lung, Right Upper Lobe, Bronchoalveolar Lavage 1: Pap-stained SurePath Liquid-based Prep; 2: H&E Cell Block Section (400X).

However, in the cell block sections, the cytologist also identified cells stranded within areas of mucin (Image 3). It may have been easy to dismiss these as macrophages, but contrary to the dusty hemosiderin-laden macrophages in this specimen, these cells had abundant vacuoles, and irregular nuclei with more prominent nucleoli (Images 4-5). The pleomorphic nuclei and larger vacuoles distinguished these cells from lipid-laden macrophages most often seen in aspiration pneumonia.

Images 3-5. Lung, Right Upper Lobe, Bronchoalveolar Lavage 3: H&E Cell Block section (100X); 4: H&E Cell Block section (400X); 5: H&E Cell Block section (600X).

The cytologist, marking the areas of interest, swiftly rendered a diagnosis of positive for malignant cells, favoring metastatic clear cell renal cell carcinoma. She enthusiastically described the case to the attending cytopathologist, who immediately ordered confirmatory immunostains on the unstained paraffin sections of the cell block.

With adequate controls, CD68 highlights the alveolar macrophages (not shown). The tumor cells show positive staining for AE1/AE3 and PAX-8 (Images 6-7), while macrophages show negative staining for AE1/AE3 and PAX-8 (Images 8-9). The tumor cells also show positive staining for vimentin (Image 10) and RCC (focal), and negative staining for TTF-1.

Images 6-10. Lung, Right Upper Lobe, Bronchoalveolar Lavage 6: AE1/AE3-positive tumor cells; 7: AE1/AE3-negative macrophages; 8: PAX-8-positive tumor cells; 9: PAX-8-negative macrophages; 10: Vimentin-positive tumor cells.

RUL BAL Final Diagnosis: Positive for malignant cells. Metastatic clear cell renal cell carcinoma.

When we attend a biopsy of an area clinically suspicious for metastatic renal cell carcinoma, we prepare ourselves for an overtly bloody sample. Even if the smears consist mainly of blood during ROSE, we rest assured that we will usually find tumor cells in the cell block sections. By obtaining a BAL of the blood-streaked RUL, the pulmonologist was able to pinpoint an area of lymphangitic spread. We often give little credit to BALs in cytology, especially when using concurrent techniques such as FNAs to sample a subcentimeter lung nodule without bulky disease. However, as seen in this case, sometimes it’s the only specimen that can help us render a diagnosis of metastatic cancer.

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

Microbiology Case Study: Middle Aged Female Undergoing Nephrolithotomy with an Encrusted Stent

Case history

A middle-aged female with a one-year history of obstructive pyelonephrosis caused by a large nephrolith was admitted for a percutaneous nephrolithotomy and cystolitholapaxy. Previous medical history was notable for hypertension, type 2 diabetes, and peripheral vascular disease. A previously placed stent and nephrostomy tube had become encrusted, necessitating surgical intervention. CT imaging demonstrated moderate hydronephrosis, progressive encrustation of the stent, and high stone burden. Pre-operative urine cultures yielded greater than 3 organisms of unclear significance. Intraoperative findings revealed a calcified stent and large renal pelvic matrix stone which was removed and sent for culture and mineral analysis.

Laboratory identification

Intraoperative specimens were sent to the microbiology laboratory for routine culture. Gram stain of the nephrolith and associated tissue was performed, revealing gram positive coryneform rods and gram positive cocci (Image 1A). Light growth of Enterococcus faecalis was observed after 24 hours, along with a heavy amount of pinpoint, gram positive rods (Image 1B). It was noted that growth was only observed on blood agar (not chocolate agar), a feature suggestive of a lipophilic species of Corynebacterium. Colonies were visible on blood agar after prolonged incubation, and the organism was determined to catalase positive. The organism produced copious amounts of urease, leading to tube positivity within 10 minutes (Image 1C). The organism was definitively identified by MALDI-TOF MS as Corynebacterium urealyticum.

Image 1. A) Representative Gram stain obtained from grind of nephrolith, and associated tissue submitted to the microbiology laboratory. A mixture of coryneform rods and Gram-positive cocci can be seen. B) Pinpoint growth of *C. urealyticum* on sheep’s blood agar media after 24 hours. Extended in incubation or the addition of lipid to the culture media is needed for more robust growth. C) Rapid urease production of *C. urealyticum*. Christiansen’s urea agar slant was inoculated with *C. urealyticum* and photographed at different timepoints post inoculation. Urease positivity can reliably be demonstrated within 10 minutes.

Discussion

Corynebacterium urealyticum is a lipophilic corynebacterial species frequently recovered from the urinary tracts of patients with renal or urological disease. While this organism is often associated with alkaline encrusted cystitis, it also plays an important role in renal stone formation. Due to its indolent and slow growing nature, the organism can be challenging to recover from routine urine cultures; thus, disease burden attributable to C. urealyticum is likely underestimated. Risk factors for disease include patients with urinary tract abnormalities, catheterization, prolonged hospitalization, history of immunocompromising conditions or renal transplant, and extended therapy with broad-spectrum antibiotics.¹ Indeed, most contemporary clinical isolates are multidrug resistant, making antibiotic therapy of C. urealyticum challenging.

C. urealyticum is a member of the colonizing flora of both the skin and the urinary tract, and its ability to adhere to the uroepithelium is believed to mediate its ability to cause disease. The organism is frequently detected in the groin of hospitalized and institutionalized patients.² As a member of the lipophilic corynebacteria, enhanced growth can be achieved through the addition of 0.1% Tween 80 to culture media. The impressive urease activity of C. urealyticum is central to pathogenesis as hydrolysis of urea in the GU tract leads to ammonia production and alkalinization (Image 1C). This, in turn, leads to saturation of the microenvironment with calcium phosphate and struvite crystallization which can result in sone formation.¹ In settings where C. urealyticum infection may be suspected (including encrusted cystitis and encrusted pyelitis), prospective discussions with the laboratory are warranted to avoid dismissal of the organism as a diphtheroid bacilli that is a normal component of the urogenital flora. This is particularly important if the laboratory does not routinely hold cultures for longer than 24 hours.

Chronic and recurrent urinary tract infections in patients of advanced age is usually the primary presentation of C. urealyticum infection. By contrast, encrusted uropathies can develop in 4-16% of patients with C. urealyticum bacteruria and are subacute-to-chronic conditions associated with urease-producing bacteria.² C. urealyticum is the principal cause of encrusted uropathies. This case represents a more complex case of encrusted disease leading to extensive nephrolith formation requiring surgical intervention. For urinary tract infections, vancomycin remains the antibiotic of choice for management in addition to removal of the impacted mucosal encrustations and urological consultation.³ C. urealyticum also exhibits near uniform susceptibility to linezolid. The excised nephrolith in this patient’s case was found to be composed of 90% struvite and 10% calcium phosphate. Following the procedure, imaging revealed no visible, residual stones in either the impacted kidney, ureter, or bladder. The patient is followed as an outpatient and continues to do well.

References

Salem, N., Salem, L., Saber, S., Ismail, G., and Bluth, M.H. Corynebacterium urealyticum: a comprehensive review of an understated organism. Infect. Drug Resist. 2015:8 129-145.
Van de Perre, E., Reichman, G., De Geyter, D., Geers, C., Wissing, K.M., and Letavernier, E. Encrusted uropathy: a Comprehensive Overview – to the Bottom of the Crust. Front. Med. 2021. 7:609024
Kim, R. and Reboli, A.C. Chapter 205: Other Coryneform Bacteria, Arcanobacterium haemolyticum, and Rhodococci in Mandell, Douglas and Bennett’s Principals and Practice of Infectious Diseases 9^th Ed. Elsevier, Philadelphia, PA. Pgs: 2532-2542.

A Culture of Safety Transparency- Three Reasons Why

Gena was working in the microbiology lab when she failed to notice a possible N. meningitidis from a cerebrospinal fluid culture. Not thinking there was any danger, she prepped the organism for identification on the analyzer, but she used a vortexer that was not in the biological safety cabinet and did not cap the tube. The next day when she saw the organism identification, she realized she had created an aerosol the previous day in the open lab when co-workers were nearby. She was afraid of getting in trouble, so she did not report the incident. Three days later, Gena was in the hospital and not expected to recover. Two other co-workers had also fallen ill with minor symptoms. After the investigation, the manager did not relay the details of the incident to all of the staff fearing that the department would get in trouble with hospital administrators. One lab employee decided to call OSHA and report what she felt were unsafe working conditions.

There are at least three reasons it is important to create a safety culture in the laboratory where all staff members feel comfortable discussing potential safety issues, incidents, and near-misses. The first reason is so that every single safety incident in the department will be reported. Even something as minor as a paper cut that occurs within the walls of the lab should be reported. A tiny wound can quickly become an infection if skin is broken while working in an area where bloodborne pathogens are present. Staff should understand why reporting is essential, and they should be aware of the follow-up procedures that are put in place by the organization. To get lab employees to realize near-miss situations may be more difficult- there needs to be education about unsafe practices and potential consequences. For example, an employee might successfully retrieve a lost specimen from a sharps container without injury, but they and others should understand the high potential consequence of that action, and it should be reported. That is a tie to discuss unknown source exposures and potential impacts.

A second reason for a culture of transparency in the lab is to help the staff know the organization s working to keep them safe and to make them comfortable enough to talk to leadership about incidents and questions they may have about them. Sometimes, if employees feel leadership is not doing a good job of this, they will report to agencies outside of the workplace. It is easy for an employee to report incidents to OSHA, and if that happens, the lab will need to do much more work for the response. When an incident occurs, being open and honest about the details, the response, and the follow up to ensure it does not happen again can go a long way toward comforting staff. Hiding information just helps to generate rumors and a feeling by some that they are not working in a place that is doing all it can to keep their employees safe. It can be difficult after certain events to tell the story. While names may not be mentioned, it is likely in many situations that staff will know who the involved parties are. That is still better than hiding information. When OSHA responds to a safety report, even if the response is written and no inspectors come on site, the incident report and the written response from the organization must be posted in the department per regulation. The employees are going to know what happened either way. It is always best to be up front about incidents and to make staff aware that their safety is important and monitored, and that issues have swift follow-up.

Safety transparency also has a third benefit- it generates an overall better safety culture for the laboratory. When staff are comfortable reporting issues without punishment or pushback, and when they see they can work with leadership to continually correct issues, safety becomes a natural part of the job. Openly reporting incidents in staff huddles, discussing routine safety fixes and improvements, and educating about near-misses are all normal in a department where the safety culture is strong. A strong safety culture means fewer incidents and fewer injuries and exposures, a goal for which all labs should strive. Once Gena realized her mistakes in the microbiology lab, she should have felt comfortable enough to report them for her safety and for the safety of others in the department. Even if she were new to the field or to the department, her leadership should have conveyed to her how vital it is to report safety incidents or potential safety issues. When staff understand that their organization cares about them and will work to protect them, even when something goes wrong, they will feel confident they work in an environment where safety transparency is the norm, and where the safety culture is strong.

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

Blunt Versus Sharp Force Injuries: A Primer on Terminology

Often in casual conversation (and even in medical records) all open skin wounds are called “lacerations,” despite the fact that this term has a fairly narrow definition. Forensic pathologists need to be very particular about using correct terminology for injuries. Different types of injuries correspond to different mechanisms of injury, which can have real consequences for an ongoing investigation. Excluding gunshot wounds, (a separate wound type that requires its own blog post), the other main categories of trauma are blunt and sharp force injuries. Blunt injuries are inflicted by dull objects, whereas sharp force injuries are inflicted by a blade or other edged object. Chop wounds, less commonly encountered in practice, have features of both.

Blunt Force Injuries

There are three cutaneous manifestations of blunt impacts. An abrasion is the equivalent of a scrape, wherein the superficial skin layer is removed. A contusion refers to a bruise, in which there is hemorrhage in the skin and subcutaneous tissue. Finally, lacerations occur when the skin and underlying tissues are crushed and tear apart from each other, resulting in an open wound. Neurovascular bundles in the subcutaneous tissue have high resistance to crushing forces and remain intact, resulting in characteristic “tissue bridging” within the wound depths. The edges of lacerations are often abraded, as well. Because blunt force injuries result from direct application of force, the object involved can impart a pattern onto the skin, or deposit potential trace evidence (hair, fibers, paint chips). It’s always important to look for patterns and try to correlate injuries with scene findings. If a patterned (or suspected patterned) injury is found at autopsy, scale photographs must be taken with a specific ruler (the ABFO ruler), to produce scale photographs for comparison if the object is found.

Deaths related to blunt force injuries can be accidental, suicidal, or homicidal in nature, and cover a wide variety of situations. Motor vehicle accidents, falls or jumps from heights, and homicidal beatings are all situations where the cause of death is blunt force injuries. Each of these situations have particular wound patterns that forensic pathologists are trained to recognize.

Sharp Force Injuries

Sharp injuries can be divided into stab wounds or incised/cutting wounds. The difference is wound dimensions: stab wounds are deeper than they are long, and incised wounds are longer than they are deep. In contrast to lacerations, sharp forces cleanly transect all tissues including neurovascular bundles, meaning stab and incised wounds show no tissue bridging. As mentioned in the first paragraph, distinguishing a laceration from a cut made with a blade can be critical. Mis-identifying a laceration as a “cut” or “stab,” or vice versa, may send law enforcement down the wrong path. Similar to blunt force, deaths by sharp force can be accidental, suicidal, or homicidal, although in my experience accidental deaths by sharp force are extremely rare.

These categories make the definitions sound clear-cut (no pun intended), but it’s important to keep in mind that in actual practice, as with everything else in medicine, unusual situations occur. ‘Chop’ wounds have features of both sharp and blunt force and are typically inflicted by a heavy object with an edge (like an axe or propeller blade). As one example, a beating with a hatchet or machete may cause sharp, blunt, or chop wounds, depending on the sharpness of the blade and what part of the weapon impacts the body. Even when an unusual object is used as a weapon, recognizing the different components of a wound and any patterns imparted on the skin can help guide an investigation.

This decedent was an ejected passenger in a motor vehicle accident. The large abrasions over the arm involve one surface of the extremity and have a faint internal pattern of parallel lines, consistent with “brush burns” or “road rash.”

*This patterned contusion on the back of a pedestrian run over by a truck matches the tire tread pattern of the vehicle*.

*This is a classic example of a laceration. Note the bands of intact tissue which span the width of the wound (the “tissue bridging”) which are characteristic of lacerations.*

A typical stab wound. Note the cleanly transected wound edges, and the absence of tissue bridging. The squared-off margin on the right, in contrast to the left side of the wound, suggests this was a single-edged blade.

-Alison Krywanczyk, MD, FASCP, is currently a Deputy Medical Examiner at the Cuyahoga County Medical Examiner’s Office.

It’s Getting Hot in Here

Each laboratory is required to create and maintain a fire prevention plan. What exactly does this plan entail? A fire prevention plan should include, at minimum, the identification of potential fire hazards in your lab, your available firefighting tools, and an action plan that outlines employees’ responsibilities during a fire or evacuation.

First, it is best to determine what fire risks are present in your labs. The best way to begin would be to inventory any flammable chemicals used and stored on-site. Some flammable materials such as alcohol can accumulate quickly, and it is necessary to know how much is stored in the department and where. The Occupational Safety and Health Administration (OSHA) mandates that quantities of flammable liquids greater than 25 gallons in a single room must be stored inside of a flammable storage cabinet (1926.152(b)(2)). The National Fire Protection Agency (specifically standards NFPA 45 and 30) takes it a bit further and focuses on limits based on total square footage in the lab. The NFPA limits the amount of flammable liquid stored outside a flammable storage cabinet to no more than 1 gallon per 100 ft², or 2 gallons per 100 ft² if you use fire safety cans. This storage limit doubles if an automatic fire suppression system is in place. The limitation of flammable materials in a concentrated area enables a fire suppression system to more easily extinguish a fire if one were to occur.

Next, look at the amount of combustible items stored around the lab. Are there several boxes of paper stacked next to photocopiers? Large amounts of combustible material in a single area can help fuel a potential fire. Are items stored too close to the ceiling? Check to see that there is at least 24 inches of clearance from the ceiling so that sprinklers are not blocked. Finally, inspect your electrical equipment. Look for daisy chains or permanently placed extension cords in the lab. As part of routine physical environmental rounding, it is best to search for these prohibited situations while also seeking out frayed cords and damaged electrical equipment.

Another component of the labs’ fire prevention is having the correct tools in place to combat a fire should one occur. The local fire authority will determine how many fire extinguishers are required in the laboratory and where they should be placed. To ensure adequate operation of this firefighting equipment, extinguishers should undergo routine checks which include annual maintenance. OSHA also requires a monthly visual inspection of all portable extinguishers (OSHA-1910.157(e)(2)). Verify that staff know the locations of their nearest fire extinguishers and that they can operate the specific types provided. Is there an automated sprinkler system in the facility? Staff should be aware of the location of fire pull alarms and have education about the alarm process (including calling any emergency numbers).

Lastly, the fire prevention plan should detail information about staff response to a fire, including fire drill and evacuation training. The safest way to evacuate is to have a predetermined evacuation route and muster (meeting) location. Staff should physically walk their full evacuation route annually all the way to their muster location and back. If this route becomes impassable, there should be an alternative evacuation route. During drills, walk one route to the muster location, then walk back via the alternate route. It is also wise to outline the expectations of staff members once they reach that muster location during the drill. If a large group evacuates at the same time, using a checklist or a buddy system can help staff keep track of who is present and who is not. Encourage your staff to stay at the muster location and not to wander off. If a supervisor is taking a roll call at the muster location, a staff member might be counted as missing if they leave to chat with a buddy in a different area. The last thing anyone wants is for a rescue worker to run into a burning building to search for a person who is not even at work that day. As the laboratory grows, so should the fire prevention plan. The addition of new equipment or a change in the current procedure warrants a review of the plan. It is recommended that fire safety policies and procedures are reviewed annually, and when changes are made, communicate that information to staff quickly. Ensuring that equipment is in place, that items are stored properly, and that staff are made ready to respond can lead to much better outcomes should a real fire occur in the laboratory.

-Jason P. Nagy, PhD, MLS(ASCP)^CMis a Lab Safety Coordinator for Sentara Healthcare, a hospital system with laboratories throughout Virginia and North Carolina. He is an experienced Technical Specialist with a background in biotechnology, molecular biology, clinical labs, and most recently, a focus in laboratory safety.

Microbiology Case Study: An Unusual Case of Herpes Reactivation

Case history

A 37-year-old female with a past medical history of psoriasis and common variable immunodeficiency disorder (CVID) presented to her dermatologist for an ulceration on her right buttock following a camping trip about 1 month ago. She thought that she had been bitten by a bug, for the lesion became extremely pruritic and painful. The patient was self-treating the area with over-the-counter antibiotic ointment and an anti-itch cream, but the symptoms persisted. At the time, the dermatologist was also treating a lower extremity dermatophyte infection, and the antibiotic cream and anti-itch cream were discontinued and replaced with clobetasol 0.05% ointment for potential allergic dermatitis. The patient returned to the dermatologist about a month later as the site was becoming increasingly inflamed and painful. The patient had also started experiencing night sweats and fever, so she was transferred and further evaluated in the emergency department. In the ED, the differential included soft tissue infection, cellulitis, or abscess of a fungal, viral, or bacterial etiology. Labs showed evidence of inflammation with an elevated ESR and CRP. A punch biopsy was performed and pathologic examination showed an ulcer bed with prominent acute inflammatory cell infiltrate and necrosis. The infected squamous epithelium showed the3 Ms findings (Molding, Margination, Multinucleation) consistent with herpetic infection (figure 1). The diagnosis was confirmed with HSV complex IHC (figure 2) and PCR testing of the lesion came back positive for HSV-2. Of note, the patient did have a history of genital herpes; however, she was not having a typical flare, and she had been treated with a 10-day course of valacyclovir 2 weeks prior to her ED visit. The gram stain showed no evidence of neutrophils, squamous epithelial cells, or organisms, but bacterial cultures came back positive for MRSA.

Figure1. H&E section showing mixed acute and chronic inflammation with squamous cells
showing herpes viral cytopathic effect (400x magnification)

Figure 2. HSV complex (HSV1 and 2) IHC staining virally infected epithelial cells (200x).

Discussion

Herpes simplex virus (HSV) is a large, double-stranded DNA virus from the Herpesviridae family.^1,2 HSV-2 is generally considered a sexually transmitted infection because it can be transmitted by contact with infected genital secretions.^2,3 The viral particles within these secretions can enter epithelial cells and begin replicating, causing the characteristic intranuclear inclusions and multinucleated giant cells that can be seen under the microscope.² When the virus infects the cells in this manner, it can also cause these infected cells to separate from each other and form grouped vesicles filled with these cell remnants.² The virus infects nerve endings and then travels backwards to the sacral ganglia, and it can remain latent there permanently, giving it the ability to recur during the infected person’s lifetime.¹ A patient can initially present with symptoms of dysuria, lymphadenopathy, fever, headaches, and myalgias, but more than half of patients may not know they have genital herpes.^1,3 Recurrences can present with symptoms of tingling, burning, itching, and pain in the nerve’s distribution pattern, similar to the pain and pruritis in our presented case.² When there is a suspected HSV infection, PCR for HSV DNA is generally the best diagnostic tool, and it is faster and more sensitive than viral culture.^1,2 A patient with known herpes infection should be treated with antivirals; however, the lesions should also self-resolve within 3 weeks if the patient is not treated.¹

One abnormality in our presented case was that the patient’s reactivation of genital herpes was on the buttocks. A repeat infection with HSV at a site other than genitalia is more common when the primary infection also occurred at a site other than the genitalia.⁴ Infections occurring at non-genital sites such as the buttocks can also occur due to self-inoculation, which may have been the case in our patient.⁴ Additionally, repeat infections with HSV in non-genital sites are more common when the initial infection was with HSV1, but our patient’s PCR showed the presence of HSV-2 DNA.⁴ One explanation for this phenomenon is that HSV-2 recurrences can occur on the buttocks due to the retrograde transport to the root ganglia in the areas that correspond to these dermatomes.⁴

Another abnormality in our presented case involves the patient’s persistent infection despite treatment with a course of valacyclovir for 10 days. Generally, an initial herpes infection self-resolves in a matter of weeks, and a recurrent episode will self-resolve in a matter of days, usually less than ten.^1,2 It is unusual that her infection persisted despite therapy, but the patient does have a medical history significant for CVID. Patients with weakened immune systems can take longer to fight off herpes infections even if they are taking antivirals.² Additionally, there is a theory that herpes buttocks infections last longer than in other regions due to the greater travel distance along the nerves as well as a higher concentration of nerve endings in this region.⁴ The patient in our case also had tissue cultures that were positive for MRSA, meaning she had a concomitant bacterial and viral infection of the buttock region, and treatment with an antiviral would not be sufficient to eradicate her coinfection.

References

1. Johnston C, Corey L. Current Concepts for Genital Herpes Simplex Virus Infection: Diagnostics and Pathogenesis of Genital Tract Shedding. Clin Microbiol Rev. Jan 2016;29(1):149-61. doi:10.1128/cmr.00043-15

2. Gupta R, Warren T, Wald A. Genital herpes. Lancet. Dec 22 2007;370(9605):2127-37. doi:10.1016/s0140-6736(07)61908-4

3. Groves MJ. Genital Herpes: A Review. Am Fam Physician. Jun 1 2016;93(11):928-34.

4. Benedetti JK, Zeh J, Selke S, Corey L. Frequency and reactivation of nongenital lesions among patients with genital herpes simplex virus. Am J Med. Mar 1995;98(3):237-42. doi:10.1016/s0002-9343(99)80369-6

-Lillian Acree is a fourth-year medical student at the Medical College of Georgia. She is interested in head and neck pathology.

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

Case Studies in Hematology: Hemoglobin S Beta Thalassemia Compound Heterozygosity in a 61 Year Old Female

A 61 year old Black woman with a diagnosis of sickle cell beta thalassemia presented to the ER with fatigue, dyspnea, and back and leg pain with some swelling in the hands and feet. Splenomegaly was noted on exam. The patient has a history of moderate to severe symptomatic anemia and is being followed by a hematologist. Her baseline Hgb is 9-10 g/dL. Her treatment plan includes Hydroxyurea, 500 mg daily, and transfusions, as needed. Her last sickle cell crisis was 2 years ago. CBC was ordered. Hgb on admission was 6.1 g/dL. Her RBC morphology showed polychromasia, target cells, sickle cells, anisocytosis, and numerous nucleated RBC forms.

The patient was admitted to the hospital. Type and crossmatch for 2 units of packed red blood cells was ordered. CT imaging was performed and revealed severe osteopenia and vertebrae deformities consistent with her history of sickle cell disease. Chest CT showed hypoinflated lungs and areas of consolidation in the lower lobes consistent with acute chest syndrome due to sickle cell beta thalassemia. She was transfused with 2 units of pRBCs and treated for sickle cell crisis. The patient remained stable and was discharged 3 days later.

Figure 1. Peripheral blood smear on admission. Patient with sickle cell/beta thalassemia shows sickle cells, target cells, nucleated red cells, anisocytosis, poikilocytosis, polychromasia. (Mercy Medical Center, Baltimore, Md)

Sickle cell anemia (HbSS) and thalassemia are the world’s most common single gene disorders. Both are inherited in an autosomal recessive manner, and result in hemolytic anemias. But what happens when you inherit one gene for sickle cell and one gene for beta-Thalassemia (β-thalassemia)?

Sickle cell disease is caused by mutations in the HBB gene that provide instructions for making beta-globin. Sickle cell anemia is a hemoglobinopathy, a qualitative defect in the structure of globin chains, resulting in the production of abnormal hemoglobin. Normal adult Hemoglobin A has 2 α chains and 2 β chains (α2β2). Hb S results from the substitution of valine for glutamic acid at position 6 of the β globin chain. The resultant Hb S has reduced solubility at low oxygen tensions. Patients with sickle cell anemia have a moderate to severe chronic hemolytic anemia with recurrent painful sickle cell crisis.

Sickle cell disease is inherited in an autosomal recessive pattern from parents who have at least one mutated gene. Anyone with a sickle cell gene can pass this gene on to their children. Sickle cell anemia (HbSS) is the homozygous expression of a sickle gene from both parents and is the world’s most common inherited hematological disease. A heterozygote inherits a sickle gene from only one parent. This person is a carrier of sickle cell (HbSs), often referred to as sickle cell trait. HbSs persons do not generally exhibit symptoms or may exhibit only a mild anemia. However, under stressful conditions, such as at high altitudes, they may experience vaso-occlusive sickle crisis.

While hemoglobinopathies are a qualitative defect due to structural changes in the normal amino acid sequence of globin, thalassemias result from an imbalance in the synthesis of the globin chains that make up the hemoglobin molecule. Thalassemias involve the rate of globin chain synthesis leading to a quantitative defect. Thalassemia is divided into α-thalassemias and β-thalassemia. α-thalassemias involve genes for the α chains on chromosome 16. In α-thalassemia, the deletions involve the α₁ and/or the α₂ globin genes and result in decreased production of α chains. β-thalassemias mainly affect β chain production. They are disorders of reduced globin chain production from the globin chain cluster on chromosome 11.

(Since this case involves a known diagnosis with a compound heterozygous state involving a β-thalassemia gene mutation, the discussion of α-thalassemia has been limited here. Watch for a case involving α-thalassemia in a future blog!)

Beta thalassemia occurs when the beta globin chains are either produced inadequately or not at all. There are many mutations in and around the β globin gene that result in decreased β chain production. Mutations that result in the complete absence of β chain production are designated as β⁰. In the most severe form of β-thalassemia the patient is homozygous β⁰/β⁰ and does not produce any β chains. Without β chains there is no Hb A (α2β2). β+ is used as the designation for any mutations of the β globin gene that cause a partial deficiency of β chains (5-30% decrease) and therefore result in a decrease in production of Hb A. The β^silentdesignation is used for carrier state gene mutations that result in only a mildly decreased β chain production. The degree of decrease in the β chain production is related to the degree of anemia and the severity of clinical disease.

Thalassemia, like sickle cell anemia, is a hereditary anemia inherited in the autosomal recessive manner. β-thalassemia is divided into categories based on clinical severity of disease. In β-thalassemia major a child inherits a copy of a β-thalassemia gene mutation from both parents. There are various mutations that cause genes with these mutations and different variants may be inherited from each parent. A person with thalassemia major may be homozygous β+/ β+, homozygous β⁰/ β⁰, or the compound heterozygous state β+/ β⁰. Hb A is only produced in patients with the β+ mutation. β-thalassemia major patients have the most severe hemolytic anemia and symptoms. β-thalassemia intermedia is characterized as homozygous β^silentor heterozygous β^silentwith β+ or β⁰ and mild to moderate disease. β-thalassemia minor, also called β-thalassemia trait or carrier state, presents with mild but asymptomatic hemolytic anemia. These patients are heterozygous with normal β globin and have slightly decreased Hb A.

In people with sickle cell disease, at least one of the beta globin is replaced with hemoglobin S. In homozygous sickle cell anemia, both beta globin subunits in hemoglobin are replaced with hemoglobin S. In compound sickle cell diseases, one beta globin is replaced with hemoglobin S and the other beta globin is replaced with a different abnormal variant. Examples of this are Hb SC disease, and Hb SD syndrome. Compound heterozygosity is the inheritance of two different mutated genes that share the same locus. If mutations that produce hemoglobin S and beta thalassemia occur together, individuals have hemoglobin S-beta thalassemia disease. (sickle cell beta-thalassemia, Hb S β thal or sickle-β-thal). Sickle cell beta thalassemia patients have hemoglobin S (α2β2^6Glu^→Val) and either β⁰ or β⁺.

When a qualitative hemoglobinopathy is inherited with a quantitative disorder of hemoglobin synthesis, the severity of the compound disorder is dependent on the β gene mutation. Patients with β⁰produce no Hb A and have moderate to severe symptoms comparable to that of Hb SS patients. β⁺ patients will produce some β chains and therefore have some Hb A and milder or no symptoms.

Figure 2. Peripheral Blood smear on day 3. Sickle cell forms, polychromasia, target cells. nucleated RBCs. (Mercy Medical Center, Baltimore, Md)

Newborn screening can diagnose β⁰-thalassemia at birth by detecting a complete absence of hemoglobin A. However, it is not possible to make a definitive diagnosis of β⁺-thalassemia in the newborn because newborns have Hb F, and the reduced amount of hemoglobin A overlaps the range for normal babies. In adults with Hb S – β thal the amount of Hb S is variable. There is some Hb A in β⁺ patients but no Hb A detected in β⁰. Hb A2 and Hb F are increased. In addition to hemoglobin electrophoresis, molecular testing may also aid in the diagnosis by identifying genetic mutations. Beta globin gene sequencing can identify beta thalassemia alleles that are caused by point mutations in the beta globin gene. As well, structural variants of the beta globin gene such as Hb S can be identified with this technique. This can lead to a better understanding and clinical management of the disease.

Case Study, continued: This patient inherited a Hb S gene from one parent and a β-thal gene from the other, resulting in sickle cell beta thalassemia. This compound heterozygosity affects red blood cells both by the production of structurally abnormal hemoglobin, and by the decreased synthesis of beta globin chains. Clinical manifestations depend on the amount of beta globin chain production. Symptoms may include anemia, vascular occlusion, acute episodes of pain, acute chest syndrome, pulmonary hypertension, sepsis, ischemic brain injury, splenic sequestration crisis and splenomegaly.

Hemoglobin electrophoresis was sent out to a reference lab and results are shown in Table 2. Based on the Hemoglobin electrophoresis, is this patient Hb S- β⁰-thal or Hb S- β⁺-thal?

Table 2. Hemoglobin pattern and concentrations of a S/betathalassemia patient

Hemoglobin electrophoresis results for Hb S beta thalassemia patients are expected to show 60-90% Hb S and 10-30% Hg F. This patient’s Hgb S at 74% is within this range. This result reflexed a sickle solubility test, which was positive. As well, the elevated Hb F and Hb A2 are consistent with this diagnosis. It was noted in the discussion above that Hb S- β⁺-thal mutations cause a decrease of 5-30% in beta chains and therefore a decrease in Hb A. This patient’s Hb S is greater than the Hb A and her Hb A concentration is 14.8%, which is consistent with this diagnosis. Hb S- β⁰mutations produce no Hb A. In this case there is some Hb A on electrophoresis but not as much as would be expected in a β⁺-thal mutation. Also, of note it that this patient was recently transfused with 2 units of pRBCs. Interpretations of hemoglobin electrophoresis assume that the patient has not been transfused in the last 3 months. The Hb A in this patient can be explained by these recent transfusions. Therefore, it can be concluded that the hemoglobin pattern and concentrations are consistent with transfusion of a Hb S beta⁰ thalassemia patient. The β⁰mutation is also consistent with this patient’s moderately severe symptomatic anemia.

References

Keohane, Elaine, et al. Rodak’s Hematology, Clinical Principles and Application, 5^th ed, Elsevier, 2016
McKenzie, Shirlyn. Clinical laboratory Hematology. Pearson Prentice Hall, 2004.
McGann PT, Nero AC, Ware RE. Clinical Features of β-Thalassemia and Sickle Cell Disease. Adv Exp Med Biol. 2017;1013:1-26. doi: 10.1007/978-1-4939-7299-9_1. PMID: 29127675.
Origa R. Beta-Thalassemia. 2000 Sep 28 [Updated 2021 Feb 4]. In: Adam MP, Mirzaa GM, Pagon RA, et al.,editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2023. Available from: https://www.ncbi.nlm.nih.gov/books/NBK1426/
Turgeon, Mary Louis. Clinical Hematology Theory and Procedures, 6^th ed, Jones and Bartlett Learning, 2017.

-Becky Socha, MS, MLS(ASCP)^CMBB^CM 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.

Drowning: A Diagnosis of Exclusion

With warmer weather approaching (or already arrived, depending on your location), it’s a good opportunity to review investigation of drowning deaths in forensic pathology. Drowning is the leading cause of deaths for children between the ages of 1 and 4 in the United States, but it can affect any age group.

Drowning is a diagnosis of exclusion – there are no pathognomonic signs of drowning, and a complete autopsy is required to rule out competing causes of death. Keeping an open mind during the investigation is the first step – discovering a body in water doesn’t automatically mean the cause of death is drowning. The body of a homicide victim may be disposed of in water as an attempt to destroy evidence, or someone may die of a cardiac arrythmia while they happen to be swimming.

The questions to answer in possible drowning deaths are much like those when faced with a body found after a fire (see “The Basics of Deaths by Fire” from February 23, 2023). Was the person alive when their body entered the water? And if they were, did they die from drowning – or did they die of another cause, and then become submerged?

Autopsies of drowned individuals commonly reveal findings supportive of, but not specific for, drowning. The lungs are typically over-expanded and edematous, and foamy fluid may be in the airways. A “foam cone” may protrude from the nostrils and/or mouth. Pulmonary effusions can be present, and the petrous ridges at the base of the skull may show red-purple discoloration due to vascular congestion and hemorrhage. The stomach and sphenoid sinus may contain large amounts of watery fluid. Wrinkling and pallor of the skin of the hands and feet (formerly called “washerwoman’s hands”) is often identified but doesn’t necessarily indicate drowning as it can occur with pre-mortem or post-mortem submersion.

A posterior neck dissection is necessary in most drownings to rule out high cervical spine injuries, which are often overlooked without this special autopsy technique. This type of trauma can be seen in divers or jumpers who strike head-first in shallow bodies of water and may cause death by itself or contribute to the decedent’s inability to self-extricate from the water. Pathologists also need to be aware that post-mortem injuries can happen as the body is passively carried by currents and bumps into rocks or other debris, known as “travel abrasions”.

The body of water is another consideration. It would be highly unlikely for a neurologically alert teenager or adult to drown in a bathtub, whereas an infant could easily drown if left unsupervised. In contrast, it may take a river or ocean with strong currents to overpower experienced swimmers. Personal medical history is important, as well – an adult with epilepsy could drown even in shallow water if they experience a seizure. The temperature of the water can also play a role. Cold water can trigger cardiac arrhythmias, contribute to fatigue of skeletal muscles, or incite hypothermia leading to a loss of consciousness. Toxicology testing is an important ancillary test in drowning deaths to provide context and may reveal intoxications that help explain someone’s inability to remove themselves from the water. Alcohol can contribute to the impairment of physical coordination and/or increase risk-taking behaviors, and has been associated with up to 70% of water recreation-associated deaths.

If the autopsy doesn’t show any indicators of drowning but reveals potentially lethal natural disease (such as severe coronary artery stenosis), then it’s likely the person died while they happened to be in the water and not because they were in the water. In every situation, though, the autopsy findings must be correlated with the decedent’s history, the results of scene investigation, and toxicology testing before a final diagnosis can be rendered. In this way, autopsies of water-associated deaths highlight the importance of context and investigation in forensic pathology.

Figure 1. A classic example of the “foam cone” seen at autopsy in instances of drowning. This finding results from marked pulmonary edema, and isn’t specific for drowning – it can be seen in many other conditions including opiate overdoses and heart disease.

Figure 2. Foamy fluid in the trachea and mainstem bronchi can be seen in any condition that causes pulmonary edema, and also is not specific for drowning.

Figure3. Wrinkling and paleness of the hands and feet is often seen in bodies recovered from water, whether or not the cause of death was actually drowning.

References

Armstrong EJ, Erskine KL. Investigation of drowning deaths: a practical review. Academic Forensic Pathology, Jan 2018.
Centers for Disease Control and Prevention, National Center for Injury Prevention and Control. Drowning Prevention. <https://www.cdc.gov/drowning/facts/index.html> Accessed 6/21/2023.

-Alison Krywanczyk, MD, FASCP, is currently a Deputy Medical Examiner at the Cuyahoga County Medical Examiner’s Office.