Category: Uncategorized

Case History

A 34 year old male presented to the emergency department (ED) with acute onset abdominal pain, nausea, vomiting, persistent fever, and chills. His physical examination at that time was consistent with appendicitis. Patient was treated with Zosyn for broad coverage. Imaging showed a normal appendix. Three days later after blood was drawn, his blood cultures flagged positive for gram negative, elongated, thin rods. Growth was determined to be Fusobacterium mortiferum by MALDI-TOF. Ampicillin/sulbactam was started and patient was given Amoxicillin/clavulanic acid for outpatient treatment. Further follow-up of the patient showed normal white blood count and normal urinalysis. Repeat blood cultures were negative.

Discussion

Fusobacteria are anaerobic, gram negative, spindle-shaped rods with pointed ends. They are part of the upper respiratory and gastrointestinal flora in humans but can cause diseases ranging from tonsillitis to septic shock.¹ Fusobacterium nucleatum and necrophorum are commonly isolated in human diseases, although other species such as Fusobacterium mortiferum, as described in our case, have occasionally been documented as a secondary cause of septicemia ² or bacteremia ¹ and in rare instances implicated in the development of thyroid abscess.³

F. nucleatum is a member of oropharyngeal flora and unsurprisingly involved in gingival and periodontal diseases.⁴ It has been also described as the most likely cause of extra-oral infections among oral anaerobes.⁵ F. nucleatum has been detected in various fetal and placental tissues associated with adverse pregnancy outcomes, such as preeclampsia, chorioamnionitis and preterm rupture of membranes.⁶ Recent studies have reported this species to be abundant in colon, esophageal carcinoma, pancreatic and breast cancers. It is associated with poor prognosis in colon, rectal, pancreatic and esophageal cancers by promoting pro-tumorigenic immune microenvironment and reduction in the number of tumor-infiltrating lymphocytes.^{7, 10} One of the proposed theories is the involvement of the Fap2 virulence factor that has been described to inhibit tumor cell clearance in colorectal cancer cells.⁸ The other commonly isolated species is F. necrophorum, which is associated with oropharyngeal infection followed by septic thrombophlebitis of the internal jugular vein with sepsis and metastatic diseases typically involving the lungs. This syndrome is known as Lemiere’s disease first described in 1936 by Andre Lemierre. F. necrophorum usually causes infection in young, otherwise healthy adults in contrast to F. nucleatum¹ which is associated more with the elderly population. According to Afra et al most of the mortality cases were due to F. nucleatum as opposed to F. necrophrum. This could be attributed to co-morbidities in elderly patients with positive F. nucleatum cultures.

Fusobacterium species can be identified using mass spectrometry MALDI-TOF. Typically, Fusobacterium species are resistant to vancomycin, but susceptible to colistin and kanamycin disk identification tests; however, F. nucleatum is susceptible to all three drugs. F. mortiferum and F. varium grow in the presence of bile. F. necrophorum shows positive indole and negative nitrate testing. Sequencing of the 16S RNA gene and 16S-23S rRNA gene spacer region can be used to determine the different species^3,9

Fusobacterium species are usually susceptible to penicillin, clindamycin, metronidazole, and chloramphenicol and resistant to macrolides. F. nucleatum and F. necrophorum may produce beta-lactamases.³ In rare cases, surgical intervention is warranted for abscess formation.

References

1. Afra K, Laupland K, Leal J, Lloyd T, Gregson D. Incidence, risk factors, and outcomes of fusobacterium species bacteremia. BMC Infect Dis. 2013;13(1). doi: 10.1186/1471-2334-13-264.
2. Prout J, Glymph R. Fusobacterium mortiferum septicemia. Clinical Microbiology Newsletter. 1985;7(4):29. doi: 10.1016/s0196-4399(85)80052-0.
3. Stavreas NP, Amanatidou CD, Hatzimanolis EG, et al. Thyroid abscess due to a mixed anaerobic infection with fusobacterium mortiferum. J Clin Microbiol. 2005;43(12):6202. doi: 10.1128/jcm.43.12.6202-6204.2005.
4. Moore WE, Moore LV. The bacteria of periodontal diseases. Periodontol 2000. 1994 Jun;5:66-77. doi: 10.1111/j.1600-0757.1994.tb00019.x. PMID: 9673163.
5. Bolstad AI, Jensen HB, Bakken V. Taxonomy, biology, and periodontal aspects of Fusobacterium nucleatum. Clin Microbiol Rev. 1996 Jan;9(1):55-71. doi: 10.1128/CMR.9.1.55. PMID: 8665477; PMCID: PMC172882.
6. Han YW. Fusobacterium nucleatum: A commensal-turned pathogen. Current Opinion in Microbiology. 2015;23:141. doi: 10.1016/j.mib.2014.11.013.
7. Alon‐maimon T, Mandelboim OO, Bachrach G. Fusobacterium nucleatum and cancer. Periodontology 2000. 2000;89(1):166. doi: 10.1111/prd.12426.
8. Umaña A, Sanders BE, Yoo CC, Casasanta MA, Udayasuryan B, Verbridge SS, Slade DJ. Utilizing Whole Fusobacterium Genomes To Identify, Correct, and Characterize Potential Virulence Protein Families. J Bacteriol. 2019 Nov 5;201(23):e00273-19. doi: 10.1128/JB.00273-19. PMID: 31501282; PMCID: PMC6832068.
9. Garcia-Carretero R, Lopez-Lomba M, Carrasco-Fernandez B, Duran-Valle MT. Clinical features and outcomes of fusobacterium species infections in a ten-year follow-up. The Journal of Critical Care Medicine. 2017;3(4):141. doi: 10.1515/jccm-2017-0029.
10. Brennan CA, Garrett WS. Fusobacterium nucleatum — symbiont, opportunist and oncobacterium. Nat Rev Microbiol. 2018;17(3):156. doi: 10.1038/s41579-018-0129-6.

-Dr. Hayk Simonyan was born and raised in Yerevan, Armenia. He attended Yerevan State Medical University after Mkhitar Heratsi where he received his doctorate degree. He did his research at The George Washington University. His studies were focused on transcription factor activation in the SFO-PVN axis that leads to cardio-metabolic changes mediated by obesity, oxidative stress, and angiotensin-II. One of his other projects included collaboration with the National Cancer Institute, working on alternative treatment for glioblastoma multiforme. His academic interests include surgical pathology and molecular. In his spare time, Hayk enjoys spending time with family, playing soccer, tennis, and skiing. Hayk is pursuing AP/CP training. 

-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.

Case History

A young man presented to the emergency department with the primary complaint of severe right hip pain persisting for 2 days. This pain began after standing uncomfortably for hours at an event. His right hip was tender to palpation with some erythema and swelling. He had no recent fall or known injury. He denied recent fever, chills, aches, constipation, diarrhea, changes in urinary habits, chest pain, and shortness of breath. An ultrasound (US) of the right hip joint showed moderate amount of effusion (Image 1). Laboratory results also showed an elevated white blood cell count (WBC).

The patient underwent incision and drainage of his right hip to relieve the swelling, and samples were collected for Gram stain and aerobic and anaerobic bacterial culture. Blood was also collected from two separate sites for culture. The blood cultures showed no growth following 5 days of incubation. Gram stain of the effusion showed 4+ WBC with no organisms seen (Image 2). The joint fluid was set up for aerobic and anaerobic bacterial culture. Anaerobic bacterial culture showed no organism growth. However, a few small, grey-ish mucoid colonies grew in the first quadrant on Chocolate agar in the aerobic culture (Image 3). Gram stain was performed on one of the colonies (Image 4). MALDI-TOF confirmed the identification of Neisseria gonorrhoeae.  

Discussion

Neisseria gonorrhoeae is a fastidious, gram negative diplococci bacteria that can grow inside neutrophils after surviving phagocytosis. It is oxidase positive and aerobic, and generally transmitted through sexual contact such as vaginal, anal, or oral sex.^1,2 After a gonococcal infection has resolved, the patient does not develop immunity to future infections from the bacteria. Reinfection is possible due to its ability to evade the immune system by varying its surface proteins, therefore making it appear novel to the immune system.³ Signs of septic arthritis include chills and fever, pain at the joint, inability to move infected joint, erythema, and swelling.⁴

Multiple factors increase the risk of septic arthritis, including a systemic blood-borne infection, IV drug use, osteoarthritis, past history of septic arthritis, rheumatoid arthritis, alcoholism, diabetes, HIV, lung or liver disorders, old age, and a suppressed immune system.⁴ Other forms of gonococcal infection are genitourinary infections, which are the most common, disseminated gonococcemia, and gonococcal ophthalmia neonatorum. Genitourinary infections can be particularly dangerous in women if left untreated, as this can lead to pelvic inflammatory disease that could result in infertility due to scarring of the fallopian tubes.^5,6 Gonococcal infections of the eyes are one of the leading cause of blindness in neonates in the United States, but can be successfully prevented through treating the mother with antibiotics before birth and administration of eye drops to the baby at birth.⁷

Identification of N. gonorrhoeae can be done using Gram stain, aerobic bacterial culture on Chocolate or Modified Thayer-Martin (MTM) agar, or nucleic acid amplification test (NAAT). Testing can be done from a urethral swab, urine sample, or sample of body fluid from the area of suspected infection.^8,9 Culture is slow with low recovery rates. In urogenital cases, where there is ample colonization of normal flora, genital flora may outgrow N. gonorrhoeae, reducing its recovery. MTM media is useful because it is a GC agar base that makes it selective for N. gonorrhoeae growth. It contains vancomycin, colistin, nystatin, and trimethoprim lactate, which suppresses growth of most other gramgnegative diplococci, gram negative bacilli, gram positive organisms, and yeast.¹⁰ The most common testing methodology for urogenital gonococcal infection is NAAT. Some FDA approved platforms also accept rectal or throat samples, however most only accept those from urogenital sources.¹¹ Also, while NAAT is a quick and sensitive diagnostic test, it has the downside of not being able to distinguish between DNA obtained from living or dead bacteria.¹²

Intravenous (IV) or intramuscular (IM) ceftriaxone is the preferred treatment choice for N. gonorrhoeae infections. Alternatively, other third generation cephalosporins can be used as well, including cefotaxmine and ceftizoxime. Typically, patients with a beta-lactam allergy have been shown to tolerate ceftriaxone, and those who cannot should undergo desensitization due to its effectiveness against this infection. A single dose of azithromycin or a prescription of doxycycline taken twice daily for a week is usually added to the regimen to cover for a potential Chlamydia trachomatis co-infection. Patients presenting with purulent arthritis should also undergo drainage, either arthroscopically or through multiple joint aspirations.^13,14

References:

1. Ryan, K. J., Ray, G., and Sherris, J. C. (2004). Sherris Medical Microbiology: An introduction to Infectious Diseases, 4^th edition. McGraw-Hill Medical.
2. Centers for Disease Control and Prevention. Gonorrhea. Available from: https://www.cdc.gov/std/gonorrhea/stdfact-gonorrhea-detailed.htm. Last updated 2021 July 22; cited on 2022 March 21.
3. Hill, S. A., Masters, T. L., and Wachter, J. Gonorrhea – an evolving disease of the new millennium. Microb Cell. 2016; 3(9): 371-89.
4. Johns Hopkins Medicine. Septic Arthritis [Internet]. 2022. Available from: https://www.hopkinsmedicine.org/health/conditions-and-diseases/arthritis/septic-arthritis. Cited 2022 March 21.
5. Levinson, W. (2014). Review of Medical Microbiology and Immunology, 13^th edition. McGraw-Hill Medical.
6. Jennings, L. K. and Krywko. D. M. Pelvic Inflammatory Disease. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021. Available from: https://www.ncbi.nlm.nih.gov/books/NBK499959/.
7. Castro Ochoa, K. J. and Mendez, M. D. Ophthalmia Neonatorum. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022. Available from: https://www.ncbi.nlm.nih.gov/books/NBK551572/.
8. Ng, L.-K. and Martin, I. E. The Laboratory Diagnosis of Neisseria gonorrhoeae. Canadian Journal of Infectious Disease and Medical Microbiology. 2005; 16: 1-11. Article ID: 323082.

9. Van Der Pol, B., Ferrero, D. V., Buck-Barrington, L., Hook 3^rd, E., Lenderman, C., Quinn, T., et al. Multicenter evaluation of the BDProbeTec ET system for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urine specimens, female endocervical swabs, and male uerthral swabs. J Clin Microbiol. 2001; 39(3): 1008–16.
10. Tankeshwar, A. Modified Thayer-Martin Agar: Preparation, Uses. Microbe Online [Internet]. Available from: https://microbeonline.com/thayer-martin-agar-composition-preparation-uses-colony-characteristics/. Cited 2022 March 29.
11. Workowski, K. A., Bachmann, L. H., Chang, P. A., Johnston, C. M., Muzny, C. A., Park, I., et al. Sexually Transmitted Infections Treatment Guidelines, 2021. MMWR Recomm Rep. 2021; 70(4): 1-187.
12. Janssen, K. J., Hoebe, C. J., Dukers-Muijrers, N. H., Eppings, L., Lucchesi, M., and Wolffs. P. F. Viability-PCR Shows That NAAT Detects a High Proportion of DNA from Non-Viable Chlamydia trachomatis. PLoS One. 2016; 11(11): e0165920.
13. Guillot, X., Delattre, E., Prati, C., and Wendling, D. Destructive septic arthritis of the sternoclavicular joint due to Neisseria gonorrhoeae. Joint Bone Spine. 2012; 79(5): 519-20. 
14. Zaia, B. E. and Soskin, P. N. Images in emergency medicine. Man with severe shoulder pain. Gonococcal arthritis of the shoulder. Ann Emerg Med. 2014; 63(5): 528-71.

-Marika L. Forsythe, MD is a PGY1 Pathology Resident at University of Chicago (NorthShore). Her academic interests include molecular diagnostics and its growing importance in the field of 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 SARS-CoV-2 testing may be dominating discussions, I wanted to highlight other important, but lesser known molecular microbiology tests, starting with BK virus.

About BKV

BK virus (BKV), a member of the Polyomaviridae family, has a tropism for uroepithelial cells and causes disease in immunosuppressed patients, particularly those who have undergone renal transplants.^1,2,3 The vast majority of immunocompetent adults are infected with BKV, with estimates up to 90%, and the bulk of cases are entirely asymptomatic.^1,3 The exact method of transmission is unknown,^3,4 but respiratory transmission is hypothesized. BKV can remain latent after initial infection and can reactivate when immunosuppressed.⁴ Intermittent asymptomatic viral shedding in urine is particularly common in pregnant individuals or elderly individuals.²

In renal transplant patients, BKV can lead to significant damage to the transplanted kidney and graft failure.¹ Polyomavirus-associated nephropathy (PVAN) can occur.² In bone marrow transplant recipients, hemorrhagic cystitis can occur as a result of this virus.² Other organ systems can be impacted although much more infrequently.⁴

The Lab’s Role in Diagnosis and Monitoring BKV

Given the profound impact on renal transplant patients in particular, these patients are routinely screened for BKV both in the blood and the urine. Importantly, BKV can be shed asymptomatically in the urine and thus correlation with BKV detection in the blood is essential. Molecular testing is the method of surveillance. There are currently no FDA approved assays for BKV so labs that perform testing use laboratory-developed tests with analyte specific reagents or research use-only kits.¹

Quantification is necessary for monitoring. As with any quantitative assay, there must be at least one negative control, one high positive control, and one low positive control included per run. All controls should fall within the linear range of the assay. To monitor for amplification inhibition, an internal control should be included for each sample.¹

We perform a BKV LDT assay here using Diasorin reagents and instrumentation. The green line is BKV target while the purple line is the internal control (IC). We run a low positive, high positive, and negative control with every run. Director review for all control and patient results is required. We use commercial BKV positive controls, which have established acceptable range that the quantification of controls must fall within for the run to be considered valid.

The Results and How They Impact Patient Care

Currently, there are no targeted treatments for BKV. In renal transplant individuals, modulation of immunosuppression is the main approach for managing BKV.^3,4 A delicate balance must be achieved as reducing immunosuppression can lead to organ rejection while high levels of BKV can cause organ failure.

References

1. 2016. 12.3 Molecular Methods for Identification of Cultured Microorganisms, Leber AL Clinical Microbiology Procedures Handbook, 4th Edition. ASM Press, Washington, DC. doi: 10.1128/9781683670438.CMPH.ch12.3
2. Gregory A. Storch and Richard S. Buller, 2019. Human Polyomaviruses, In: Carroll KC, Pfaller MA Manual of Clinical Microbiology, 12th Edition. ASM Press, Washington, DC. doi: 10.1128/9781683670438.MCM.ch108
3. Furmaga J, Kowalczyk M, Zapolski T, et al. BK Polyomavirus-Biology, Genomic Variation and Diagnosis. Viruses. 2021;13(8):1502. Published 2021 Jul 30. doi:10.3390/v13081502
4. Mark D. Reploeg, Gregory A. Storch, David B. Clifford, BK Virus: A Clinical Review, Clinical Infectious Diseases, Volume 33, Issue 2, 15 July 2001, Pages 191–202, https://doi.org/10.1086/321813

-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.

Case History

An 18 year old healthy female presented to the emergency department of a tertiary care hospital in Minnesota for headache, vomiting, and sore throat. She did not have any significant past medical history. Due to meningitis concerns, lumbar puncture and head computed tomography (CT) imaging were performed. The CT scan showed an accumulation of fluid in the posterior right frontal sinus with scattered mucosal thickening. However, her cerebrospinal fluid (CSF) profile was insignificant, with normal protein and glucose levels. CSF culture was ordered, and two sets of blood cultures were drawn. 

Based on the examination and presenting symptoms, pharyngitis was suspected, and she was discharged with Amoxicillin (500mg Q6H for five days). However, her strep throat screening returned negative. Her blood culture was negative. CSF culture was also negative. Cryptococcal antigen and Enterovirus PCR were performed; however, both results were negative.

She returned to the ED two days later for a worsening headache and newly developed photophobia. Additional history revealed that she went swimming in a lake two weeks prior to her first presentation at the ED. Her CSF was sent for the Ova and Parasite (O&P) exam for suspicious parasitic meningitis. The CSF O&P test was negative. CSF PCR for amoeba was also performed at a reference laboratory, and the results came back positive with Balamuthia mandrillaris. The patient was then given flucytosine, fluconazole, and azithromycin. 

Discussion

Balamuthia mandrillaris belongs to a group of free-living amoebae, including Acanthamoeba species and Naegleriafowleri, that cause fatal encephalitis.¹ Balamuthia mandrillaris is the only known species of the genus Balamuthia that causes infections in humans. Encephalitis caused by B. mandrillaris is known as granulomatous amoebic encephalitis (GAE). GAE is characterized as a subacute to a chronic infection that can last several months to years.² GAE differs from primary amoebic meningoencephalitis (PAM) caused by Naegleria fowleri, which typically causes an acute onset lasting a few days. 

While ecological niches of B. mandrillaris are not well understood, they have been reported to be isolated from dust, soil, and water.^r Both trophozoite and cyst forms can enter the body through the nasal passage or ulcerated/broken skin; however, the trophozoite stage causes associated disease manifestation and represents a diagnostic stage.² 

Brain-eating amoebas are traditionally difficult to diagnose. Hematology and chemistry profiles of CSF of affected individuals are generally unremarkable, although, sometimes, increased monocytes and lymphocytes, along with increased protein levels, are seen in some cases of GAE.³ 

The most common method of laboratory diagnosis of B. mandrillaris is a microscopic examination of CSF wet mount (Figure 1) or via immunohistochemical staining of CSF or brain biopsy.¹ With advancements in technology, species-specific nucleic acid amplification tests (NAAT) can be performed to diagnose B. mandrillaris infection accurately. However, there is no commercially available NAAT for the free-living amoeba. Only very few laboratories, such as State departments of health laboratories, Centers for Disease Control (CDC) and Prevention, or commercial reference laboratories, develop these tests as a laboratory-developed test (LDT). Histological assessment of biopsies from brain lesions may reveal tumor-like appearance or perivascular monocytic necrosis of affected areas.¹ While there have been significant technological advancements, the prognosis stays at less than a 5% survival rate,⁶ with only roughly 25% of cases diagnosed antemortem. One possible reason for delayed laboratory diagnosis is the challenges in performing the microscopic examination in clinical microbiology laboratories since it requires expertise for accurate identification of the organism. Additionally, most clinical microbiology laboratories do not readily have an in-house LDT for free-living amoeba NAAT. Therefore, the turnaround time for diagnosing B. mandrillaris or any free-living amoeba is typically longer when specimens have to be sent out to reference laboratories. 

Diagnosis of B. mandrillaris encephalitis solely based on clinical symptoms is often challenging due to similar presentation in other causes of infectious encephalitis. B. mandrillaris can affect both immunocompetent and immunocompromised individuals.^1,3,4 The first B. mandrillaris case was reported in a deceased baboon in the San Diego Zoo in 1986.¹  The majority of patients were diagnosed postmortem.¹ While most B. mandrillaris infections are actively acquired through nasal passages or skin penetration, rare post-mortem cases of passive transfer of the organism from organ transplantation have been reported.⁶ With technological advancement, there have been successes in pre-mortem diagnoses in recent years.^1,4 According to the known cases, individuals of Latin American origin are more likely to contract the disease; it is unknown if it is due to increased exposure or a genetic predisposition.¹  Similar to other free-living amoebae, B. mandrillaris can be generally found in warmer climates or tropical regions. Of approximately two hundred cases reported worldwide, about 34 were reported in Latin America, from Mexico to Brazil, while some were from Japan, New Zealand, England, and other European countries. The Southwestern United States also contributes 30 cases, mostly in Arizona, Texas, and California.¹  In the United States, there have only been 109 cases directly reported to CDC from 1974 to 2016.^2,7  We believe that this is the first case of Balamuthia reported in Minnesota. The number of exact cases would be difficult to be determined due to misdiagnosis and rare occurrence of the disease or cases not reported to CDC or the state department of health. 

While investigational drugs for B. mandrillaris GAE are in development, combination therapy of flucytosine, fluconazole, pentamidine, and azithromycin or clarithromycin has shown successes.² Our patient was successfully treated with flucytosine, fluconazole, and azithromycin. 

References

1. Matin A, Siddiqui R, Jayasekera S, Khan NA. Increasing importance of Balamuthia mandrillaris. Clin Microbiol Rev. 2008 Jul;21(3):435-48. doi: 10.1128/CMR.00056-07. PMID: 18625680; PMCID: PMC2493082. 
2. Centers for Disease Control and Prevention. (2019, August 23). CDC – Dpdx – free Living Amebic Infections. Centers for Disease Control and Prevention. https://www.cdc.gov/dpdx/freelivingamebic/index.html.
3. Kofman A, Guarner J. Free Living Amoebic Infections: Review. J Clin Microbiol. 2021 Jun 16:JCM0022821. doi: 10.1128/JCM.00228-21. Epub ahead of print. PMID: 34133896.
4. Pietrucha-Dilanchian, P., Chan, J. C., Castellano-Sanchez, A., Hirzel, A., Laowansiri, P., Tuda, C., Visvesvara, G. S., Qvarnstrom, Y., & Ratzan, K. R. (2011). Balamuthia mandrillaris And Acanthamoeba Amebic Encephalitis With Neurotoxoplasmosis Coinfection in a patient with Advanced HIV Infection. Journal of Clinical Microbiology, 50(3), 1128–1131.
5. Ong TYY, Khan NA, Siddiqui R. 2017. Brain-eating amoebae: predilection sites in the brain and disease outcome. J Clin Microbiol 55:1989 –1997. https://doi.org/10.1128/JCM. 02300-16.
6. Centers for Disease Control and Prevention. 2011. Balamuthia mandrillaris transmitted through organ transplantation—Mississippi, 2009. Am J Trans-plant 11:173–176. https://doi.org/10.1111/j.1600-6143.2010.03395_1.x.
7. Jennifer R Cope, Janet Landa, Hannah Nethercut, Sarah A Collier, Carol Glaser, Melanie Moser, Raghuveer Puttagunta, Jonathan S Yoder, Ibne K Ali, Sharon L Roy, The Epidemiology and Clinical Features of Balamuthia mandrillaris Disease in the United States, 1974–2016, Clinical Infectious Diseases, Volume 68, Issue 11, 1 June 2019, Pages 1815–1822, https://doi.org/10.1093/cid/ciy813

-Alejandro Soto, MLS (ASCP)^CM is a junior medical technologist who is passionate about clinical microbiology.

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

Case Description

A 62 year old male with unknown past medical history was dropped off at the emergency department by EMS after being found altered with concern for IV drug use. On presentation he was febrile to 104.5^o F, tachycardic, and although he was initially responsive, his mental status deteriorated. Labs were drawn and broad-spectrum antibiotic coverage with vancomycin, cefepime, and metronidazole was initiated in the ED. He then had a tonic-clonic seizure event and was given intravenous levetiracetam. A CT brain showed a right inferior temporal lobe lesion, initially interpreted as likely glioblastoma multiforme, causing subfalcine and uncal herniation. MRI revealed a ring-enhancing mass measuring 3 cm x 3 cm x 3 cm in the right temporal lobe with significant surrounding edema. CT of the temporal bones also revealed right mastoiditis (Figures 1 and 2).

Neurosurgery evacuated 17 mL of fluid from the mass and a ventricular drain was placed. Gram stain of the evacuated fluid identified many white blood cells and few gram-positive cocci in pairs, chains and clusters (Figure 3). Postoperatively, the patient was mechanically ventilated and medicines were used to support his blood pressure in the ICU. Broad-spectrum antibiotics were continued for CNS penetration and activity against possible oral flora.

An aerobic culture of drained contents from the brain ultimately grew characteristic alpha-hemolytic colonies with central umbilication (Figure 4) which were subsequently identified by MALDI-TOF and optochin disk as Streptococcus pneumoniae. Admission blood cultures also grew Streptococcus pneumoniae with characteristic “bullet-” or “lancet-shaped” gram-positive cocci in pairs on Gram stain. Fungal and acid-fast bacillus cultures had no growth. Following susceptibility testing; antibiotic coverage was narrowed to IV Penicillin G.

The patient remained unresponsive and required continued intensive medical support. Although blood cultures were sterilized, he continued to have fevers and persistent leukocytosis. Gram stain of ventricular drainage re-demonstrated gram positive diplococci. The patient was transitioned to comfort care and expired on day 5 of hospitalization, the cause of death was sepsis.

Discussion

This case of a brain abscess demonstrates an unusual intracranial complication of Streptococcus pneumoniae. S. pneumoniae (or pneumococcus) is a commensal of the upper respiratory tract (URT) and important opportunistic pathogen. Up to 65% of children and less than 10% of adults are colonized by S. pneumoniae. Dissemination of S. pneumoniae beyond its niche in the nasal mucosa leads to a spectrum of disease including lobar pneumonia, meningitis, sepsis, sinus infections and middle ear infections.¹ Local dissemination of S. pneumoniae to the central nervous system (CNS) is the most common intracranial complication of otitis media and mastoiditis. These patients can present with fulminant “otogenic” meningitis. About a third of these cases require myringotomy or mastoidectomy.² Focal parenchymal brain infection by pneumococcus, however, is uncommon.

This patient presented with signs of mass effect due to a large temporal lobe abscess warranting emergent neurosurgery. Broadly, focal parenchymal brain infections arise either by hematogenous dissemination of organisms or contiguous spread from an adjacent infection. The age, immune status, and any underlying disease present in the patient help predict the pathogen. Brain imaging is also helpful. Hematogenous spread, usually from endocarditis, tends to produce multiple lesions at the grey-white matter junction,³ while direct seeding causes solitary lesions.^4,5 In this older patient with a relatively intact immune system and a possible history of intravenous drug use, hematogenous spread of bacteria was considered. However, a large single lesion in the temporal lobe with a plausible adjacent nidus (opacified mastoid air cells) is most consistent with contiguous spread.

A wide range of organisms should be considered when evaluating brain abscesses, though S. pneumoniae is a relatively rare culprit. A meta-analysis of 9,699 patients with brain abscesses found that S. pneumoniae was isolated from only 2.4%.⁶ The most common organisms were streptococci of the viridans group (34%) and Staphylococcus spp., most commonly S. aureus (18%). Even among patients with otogenic intracranial abscesses, S. pneumoniae is rarely implicated. Interestingly, the pathogen most frequently isolated from otogenic brain abscesses is Proteus mirabilis. ^7,8

Once S. pneumoniae was identified, susceptibility testing was required to rule out acquired resistance to beta lactam and cephalosporin antibiotics, which is mediated by altered penicillin-binding proteins (PBPs).⁹ A more stringent susceptibility minimal inhibitory concentration (MIC) breakpoint applies to S. pneumoniae meningitis than other infections to account for drug distribution into the CNS.¹⁰ The hospital antibiogram reports that 97% of S. pneumoniae isolates are susceptible to Penicillin at MICs acceptable for treating non-meningitis infection but only 53% are susceptible at MICs for meningitis. Furthermore, 3.3% of all strains reported in the United States between 2001 and 2005 were also significantly resistant to ceftriaxone.¹¹ This patient was covered by broad spectrum antibiotics until susceptibility testing demonstrated sensitivity to both penicillin and ceftriaxone.

References

1          Weiser, J. N., Ferreira, D. M. & Paton, J. C. Streptococcus pneumoniae: transmission, colonization and invasion. Nat Rev Microbiol 16, 355-367, doi:10.1038/s41579-018-0001-8 (2018).

2          Kaplan, D. M., Gluck, O., Kraus, M., Slovik, Y. & Juwad, H. Acute bacterial meningitis caused by acute otitis media in adults: A series of 12 patients. Ear Nose Throat J 96, 20-28 (2017).

3          Bakshi, R. et al. Cranial magnetic resonance imaging findings in bacterial endocarditis: the neuroimaging spectrum of septic brain embolization demonstrated in twelve patients. J Neuroimaging 9, 78-84, doi:10.1111/jon19999278 (1999).

4          Brouwer, M. C., Tunkel, A. R., McKhann, G. M., 2nd & van de Beek, D. Brain abscess. N Engl J Med 371, 447-456, doi:10.1056/NEJMra1301635 (2014).

5          Miller, J. M. et al. A Guide to Utilization of the Microbiology Laboratory for Diagnosis of Infectious Diseases: 2018 Update by the Infectious Diseases Society of America and the American Society for Microbiology. Clin Infect Dis 67, e1-e94, doi:10.1093/cid/ciy381 (2018).

6          Brouwer, M. C., Coutinho, J. M. & van de Beek, D. Clinical characteristics and outcome of brain abscess: systematic review and meta-analysis. Neurology 82, 806-813, doi:10.1212/WNL.0000000000000172 (2014).

7          Duarte, M. J. et al. Otogenic brain abscesses: A systematic review. Laryngoscope Investig Otolaryngol 3, 198-208, doi:10.1002/lio2.150 (2018).

8          Kangsanarak, J., Fooanant, S., Ruckphaopunt, K., Navacharoen, N. & Teotrakul, S. Extracranial and intracranial complications of suppurative otitis media. Report of 102 cases. J Laryngol Otol 107, 999-1004, doi:10.1017/s0022215100125095 (1993).

9          Chen, L. F., Chopra, T. & Kaye, K. S. Pathogens resistant to antibacterial agents. Infect Dis Clin North Am 23, 817-845, vii, doi:10.1016/j.idc.2009.06.002 (2009).

10        Weinstein, M. P., Klugman, K. P. & Jones, R. N. Rationale for revised penicillin susceptibility breakpoints versus Streptococcus pneumoniae: coping with antimicrobial susceptibility in an era of resistance. Clin Infect Dis 48, 1596-1600, doi:10.1086/598975 (2009).

11        Sahm, D. F. et al. Tracking resistance among bacterial respiratory tract pathogens: summary of findings of the TRUST Surveillance Initiative, 2001-2005. Postgrad Med 120, 8-15, doi:10.3810/pgm.2008.09.suppl52.279 (2008).

–Miles Black, Ph.D. is a fourth-year medical student in the Medical Scientist Training Program at UT Southwestern Medical Center. His background is in enzyme biochemistry and Legionella pathogenesis.

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

–Dominick Cavuoti, D.O. is a professor of Pathology at UT Southwestern Medical Center who specializes in Medical Microbiology, ID Pathology and Cytology.

-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.

Pre-analytic factors contribute to over 70% of laboratory errors. The most common examples include missing test request, wrong/ missing ID, contamination from collection site, hemolyzed/ clotted/ insufficient sample, wrong container or wrong transport conditions. 

In our NGS lab, pre-analytic factors rarely arise. Sometimes an incorrect slide was sent, so then the tumor and germline DNA don’t match. We do perform many rounds of PCR (total 30-35 cycles), so contamination of amplified PCR products could be a concern. Usually, our volume is low enough that contamination isn’t a significant issue and not one we have noticed.

Multiple mutations- A COVID-19 Hybrid?

In a batch of COVID-19 whole genome sequencing samples from early July, I noticed some interesting mutations. Mutations from the Alpha and Delta variants were showing up in the same specimen. At first I thought this could mean a hybrid virus. Another possibility was co-infection in the same patient. And lastly, the specimen could just be contaminated. The best way to resolve this issue is to check the “phase” of the viral variant sequencing reads. The phase refers to the single pieces of DNA that are read by the sequencer. They are normally short (75-150bp in length), but if you can see whether the variants occur on the same read or only on different reads it rules out the possibility of a hybrid virus.

Determining the difference between a hybrid or 2 viral genomes

The difficulty is finding a location within both the Alpha and Delta variants that have mutations close to each other. One region exists near a deletion at amino acid 144 (Alpha) and amino acids 157-158 (Delta).

Tracing the source of the issue

As there were no overlaping reads with the 3 b.p. and 6 b.p. deletion, we concluded that this finding arose from 2 distinct viral genomes. As to whether the person was infected with both Alpha and Delta, we looked to the rest of our 96 well plate. A characteristic muation in Alpha spike protein is N501Y (it confers increased binding to ACE2R and increased infectivity). This mutation was found in several specimens of the Delta lineage, but in this case there was a much lower frequency of this variant compared to total reads. Also, several of the cases had high CT values to start with. Many of the contaminated samples also were near a variant that mapped strongly to Alpha and had a lower Ct value (CT=25). Mapping the location of the specmens to the plate showed close proximity to the authentic Alpha variant.

Luckily, before all WGS testing, we perform a targeted PCR, which I’ve mentioned in previous blog posts. This showed that only one sample (well F3) was B.1.1.7, and the rest were Delta variants. Thus we concluded we had experienced a case of contamination.

Since this time, we’ve had issues with the negative control having borderline positive levels of sequencing data that maps to Delta (now 100% of all cases and with low CT values). This is apparently a problem at multiple labs, but one that we are trying to address by looking at several root causes.

Pre-analytic concerns: Contamination sources

COVID-19 viral genome sequencing has found issues of contamination in several circumstances. We attribute this to a few reasons:

• High viral load of some specimens (especially Delta)
• Thin plastic covers that pop off easily, especially when taken out of the freezer
• And a large volume of specimens being processed.

For an example of the plastic cover issue, you can see this picture below where the cold temperature of -80C causes the plastic film adhesive to come off quickly as it is removed from cold storage. It makes popping sounds and could aerosolize viral particles to other wells in the plate.

We now use aluminum PCR plate covers that do not come off with freeze/ thaw transitions. Furthermore, we use a multi-channel pipette that pieces the cover to withdraw individual samples without exposing them to other wells.

We have also implemented bioinformatic QC metric cut-offs to determine where a cut-off for negative specimens should be. We decided on an ambiguity score <0.5. The results were consistent with previous findings where lineages could be assigned at a CT >30, but sometimes they failed as CT values increased. Negative controls were assigned a CT of 40 and all fell below the 0.5 cut-off. This has been a useful metric to be sure we are providing high quality results.

Concluding remarks

Pre-analytic factors impact every part of testing and as COVID-19 sequencing has shown, even the NGS lab tests are not immune to these challenges.

The targeted PCR test helped flag/ resolve several of the issues as they arose.

COVID-19 sequencing is still for research/ epidemiologic purposes and demonstrates the importance of rigorous clinical validations to mitigate issues such as carry-over.

References Lippi G, Chance JJ, Church S, Dazzi P, Fontana R, Giavarina D, et al. Preanalytical quality improvement: from dream to reality. Clin Chem Lab Med. 2011;49:1113–26.

-Jeff SoRelle, MD is Assistant Professor of Pathology at the University of Texas Southwestern Medical Center in Dallas, TX working in the Next Generation Sequencing lab. His research interests include the genetics of allergy, COVID-19 variant sequencing, and lab medicine of transgender healthcare. Follow him on Twitter @Jeff_SoRelle.

Case History

A woman in her 40s presented to her primary care physician in summer 2020 with mild abdominal pain, diarrhea, nausea, and headache. She experienced loose bowel movements 3 – 4 times per day for the past 18 days. She denied bloody stools, travel, consumption of raw or undercooked meats or unpasteurized dairy, contact with animals, or recent antibiotic use. SARS-CoV-2 PCR was negative. A stool sample was collected and sent for an enteric panel PCR (Salmonella, Shigella, Campylobacter, and Shiga toxin), a bacterial stool culture (Aeromonas, Plesiomonas, and Vibrio), and an ova and parasite (O&P) exam with a request to perform a modified acid-fast stain. While the enteric panel and bacterial stool culture were negative, the following organism was observed on the modified acid-fast stain (Image 1). This organism measured approximately 9 µm, was variably modified acid-fast, and had a wrinkled-cellophane appearance. The organism was identified as a Cyclospora cayetanensis oocyst. The patient later shared that she had consumed a bagged salad mix that was implicated in the ongoing Cyclospora outbreak.

Cyclospora cayetanensis

Cyclospora cayetanensis, a coccidian protozoan, is transmitted through ingestion of food or water contaminated with infectious oocysts. While infected humans shed oocysts in their stool, these oocysts are unsporulated and non-infectious at the time of excretion. In order to sporulate and become infectious, these excreted oocysts must incubate in the environment for 7 – 15 days post-excretion. Due to the required incubation post-excretion, direct fecal-oral transmission cannot occur.

Endemic areas include Central and South America, Middle East, South East Asia, and the Indian subcontinent. In non-endemic areas, travelers make up a large proportion of cases. Local outbreaks in non-endemic areas are often due to contaminated food sources. Most commonly the source of these outbreaks arise from consumption of raw fruits and vegetables that are difficult to thoroughly clean. These include leafy green vegetables (salad mixes, lettuce), herbs (basil, cilantro), and raspberries. Moreover, Cyclospora is resistant to many disinfectants used in the food industry. As exposure to this parasite is through contaminated food and water, infected patients are also at risk for other food and waterborne parasites including Cryptosporidium.

Once infectious oocysts are ingested, symptoms are typically observed after a one week incubation. Clinical presentation of Cyclospora infection includes diarrhea, nausea, fatigue, low grade fever, and weight loss. Although Cyclospora causes infections in both immunocompromised and immunocompetent individuals, symptoms may be severe and prolonged in immunocompromised patients, particularly in HIV and AIDS patients. Children and elderly individuals are also at higher risk for severe disease. Trimethoprim-sulfamethoxazole is the standard treatment. If untreated, symptoms can last for 10 – 12 weeks and may exhibit a relapsing pattern.

Stool samples should be submitted to the clinical microbiology laboratory for microscopic and/or molecular studies. To increase recovery of the organism during intermittent or low burden shedding, multiple stool specimens should be submitted over 2 -3 days. When viewed under a UV fluorescent microscope, Cyclospora oocysts autofluoresce and appear blue or green. While safranin-based stains or UV fluorescent microscopic examination can be used, modified acid-fast staining is commonly performed for the microscopic identification of Cyclospora. Cyclospora oocysts are modified acid-fast variable and measure 8 – 10 µm in diameter, unlike Cryptosporidium oocysts which are modified acid-fast positive and measure 4 – 6 µm in diameter. It is important to alert the clinical microbiology lab if suspecting Cyclospora as stains used in routine O&P exam, including trichrome stains, are not effective in highlighting Cyclospora. Although lab developed tests and FDA cleared multiplex gastrointestinal pathogen panels including Cyclospora are available, molecular assays are not yet routinely used for identification of Cyclospora due limited widespread availability.

References

1. Almeria S, Cinar HN, Dubey JP. Cyclospora cayetanensis and Cyclosporiasis: An Update. Microorganisms. 2019;7(9):317. Published 2019 Sep 4. doi:10.3390/microorganisms7090317
2. Hadjilouka A, Tsaltas D. Cyclospora Cayetanensis-Major Outbreaks from Ready to Eat Fresh Fruits and Vegetables. Foods. 2020;9(11):1703. Published 2020 Nov 20. doi:10.3390/foods9111703
3. Ortega YR, Sanchez R. Update on Cyclospora cayetanensis, a food-borne and waterborne parasite. Clin Microbiol Rev. 2010;23(1):218-234. doi:10.1128/CMR.00026-09
4. Garcia LS. Diagnostic Medical Parasitology. 6^th Edition. 2016.
5. Casillas SM, Bennett C, Straily A. Notes from the Field: Multiple Cyclosporiasis Outbreaks — United States, 2018. MMWR Morb Mortal Wkly Rep 2018;67:1101–1102. DOI: http://dx.doi.org/10.15585/mmwr.mm6739a6

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.

On which side of the aisle do you stand regarding the subject of change? Things change, or things never change? The only constant is change, or it’s always the same old thing? When it comes to the laboratory safety culture, there are some generally-accepted thoughts; change is difficult, change is slow, and change takes persistence and patience.

I’ve heard other things too- people hate change, or people like change as long as they get to be in charge of it. I do believe most of us like change. After all, we change our clothes, we re-arrange our furniture, we remodel a room in our home. It can be exciting, but the tables seem to turn if it’s a change that is forced upon us or that was not our decision (Think about all of the changes the pandemic forced upon us last year!). Changing your lab safety culture for the better can be difficult, but it can be done. First, however, you need to know the current culture and goings-on in your lab in order to be able to make a difference.

There are specific ways to determine the safety culture in your lab. An experienced safety professional can do it fairly quickly. For others, especially those who serve in multiple capacities (you know who you are- you’re in charge of lab safety but you’re also the lab manager, or the quality coordinator, or the POCT coordinator) – for you assessing the culture can be difficult, even with years of experience- because you have so many other things on your plate. That can hinder your ability to make quick assessments, but it will not hinder you completely from being able to make a true safety assessment.

To make an assessment you need to use three specific tools that you likely have at your disposal. These tools may come in a variety of forms.

Those who have followed my work for some time know about the tool “Safety Eyes.” This is a safety assessment tool I believe to be a “superpower” that we all have and need to develop. It is so powerful, in fact, that a developed user can make a fairly good and accurate safety assessment with a quick glance into the department. Performing a lab safety audit is also a very valuable tool that can give you much information about the department’s culture. Perform a complete audit at least annually, and follow-up on the results. Otherwise, you have wasted your time and resources.

A second important safety culture gauge is the use of a written or electronic safety culture assessment. You may be able to tell what’s going on visually and physically by the evidence of your eyes and safety audits, but this tool is a way to actually get into the heads of your staff. What do they think of the culture? What is their opinion of it? What do they think needs improvement, and how would they suggest making those changes? A safety culture assessment can be given to everyone, or it can be used for specific lab groups. Survey the lab staff, survey those responsible for safety, or survey lab leadership. You should perform a lab safety culture assessment at least annually, but it can be done more often as needed.

Lastly, you can use laboratory data that you already collect to see the current state of safety in the department. Analyzing the data you collect about the injuries, accidents and exposures in your laboratory can be very eye-opening, and if you share the data as safety education, you may be able to lower the number of these types of incidents. Look at the chemical and biological spills in the lab. Analyze how they happened and how to prevent a re-occurrence. If you’re the quality coordinator for your lab or system, you know about root cause and common cause analyses. The incidents that occur in the lab that generate a root cause investigation may not always be about lab safety, but it’s possible that investigations show safety is a key factor, and those results should be reviewed with the safety person in the lab.

There is much fact-gathering in the laboratory setting, even regarding the topic of safety. However, all of that data becomes worthless if there is no action taken with it. Audits, injury data, spill information – it can be very valuable information and it can all be used as tools to help you truly change your lab safety culture. If you use them properly, you can make a change, you can make a difference, and you might just end up on the correct side of the change aisle!

–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.

Case Description

A 60 year old Hispanic male with a past medical history significant for chronic pancreatitis, hypertension and cirrhosis was admitted with decompensated cirrhosis. He underwent paracentesis for ascites and subsequently developed a hematoma as a complication of the procedure which required embolization. During his 12-day long hospital stay, he also developed hypoxia due to volume overload that improved with diuresis. A Foley catheter was placed during his hospital stay which was removed prior to discharge. Weeks later, at a follow up appointment with urology, he complained of dysuria, very little urine during voiding and the sensation of incomplete bladder emptying. A clean catch urine culture was performed and grew >100,000 colonies of Escherichia coli. As shown in Table 1, the isolate was resistant to multiple classes of antibiotics including penicillins, cephalosporins, fluoroquinolones, one aminoglycoside (Tobramycin), Trimethoprim/Sulfamethoxazole, aztreonam and carbapenems (Ertapenem/Meropenem) making this isolate multi-drug resistant (MDR). Because of the resistance profile to the carbapenems, molecular testing for carbapenemase genes was performed and the New Delhi metallo-beta-lactamase (NDM-1) gene was detected. The patient was treated with nitrofurantoin for his symptomatic urinary tract infection (UTI).

Discussion

Escherichia coli is a gram negative, motile bacillus that is a normal constituent of the gastrointestinal tract and is one of the most common causes of uncomplicated UTI. Antimicrobial susceptibilities are nearly always performed because the isolates of E. coli can vary in resistance. E. coli do not have any intrinsic resistance to antibiotics other than penicillin; however, they can acquire resistance through numerous mechanisms including structural mutations and plasmid-borne genes that encode enzymes to various classes of antibiotics. One such plasmid-encoded enzyme is the NDM, which was identified in our patient’s isolate. Its resistance is the result of bacterial synthesis of a carbapenemase that deactivates carbapenems by breaking down the beta-lactam ring.¹ In the United States, K. pneumoniae carbapenemase (KPC) is the most common, but other types carbapenemase enzymes have also been reported.^1,2 NDM is uncommonly isolated in E. coli; it is more often identified in other gram negative bacteria including MDR Pseudomonas aeruginosa or Acinetobacter baumannii complex, which can cause, among other things, devastating nosocomial infections within a healthcare setting. Because these enzymes are on mobile elements, a patient can be colonized with one bacterial strain that carries the plasmid with the carbapenemase on it and transfer a copy of that plasmid to another bacterial strain, thereby conferring new carbapenem resistance to the new bacterium (e.g., P. aeruginosa with the NDM on a plasmid shares that plasmid with an E. coli). Carbapenem resistant Enterobacteriaceae (CRE) are of great importance in healthcare. Carbapenem resistance mediated by enzyme activity (e.g., KPC, NDM, OXA-48, etc), typically confers resistance to all beta lactams. Interestingly, NDM enzymes typically do not destroy aztreonam, a monobactam;³ however, it is common for bacteria to have multiple resistance genes, so NDM carrying strains can be resistant to aztreonam. Although these CRE isolates can cause significant morbidity and mortality when found in clinical samples including sputum or blood, luckily for our patient, he had an uncomplicated UTI and nitrofurantoin was susceptible.

-Limin Yang is a PGY-1 resident in Anatomic and Clinical Pathology at University of Texas Southwestern. She has varied interests including anatomic pathology specialties.

-Dominick Cavuoti is a professor of Anatomic and Clinical Pathology at UT Southwestern and active faculty on both Microbiology and Cytology services.

-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.