Tag: microbiology

Case History

A 73 year old male with a complex medical history, including type 1 diabetes, coronary artery disease, atrial fibrillation, hypothyroidism, hyperlipidemia, glaucoma, previous coronary artery bypass grafting (CABG), transcatheter aortic valve replacement (TAVR), and an implanted cardioverter-defibrillator (ICD), presented with bilateral persistent paraspinal neck pain, shaking chills, and myalgias. The initial evaluation involved broad laboratory and imaging work-up, which showed mildly elevated CRP and erythrocyte sedimentation rate (ESR). Chest X-ray, CTA head/neck, and CT thorax were normal. The patient was discharged with symptoms resolved using Tylenol and Valium, and instructions for follow-up care were provided. Two days later, the patient was recalled to the ED after blood cultures were positive for gram positive cocci in pairs and chains. The patient, asymptomatic at the time, was informed of the findings and agreed to return for further evaluation.

The patient had undergone a tooth extraction followed by implant placement one month prior, raising concerns for endocarditis due to prosthetic valve and pacemaker. He was transferred to the cardiology service, and repeat cultures were ordered. Initially afebrile, he later developed a fever (101.7°F), and his condition was complicated by diabetic ketoacidosis (DKA) and septic shock requiring ICU admission. The patient was treated with IV Vancomycin 1.5 g q24h and recommended to have repeat blood cultures until clearance. The discharge diagnosis was bacteremia caused by Abiotrophia defectiva, with a possible prosthetic valve endocarditis. A six-week course of Vancomycin was prescribed.

Discussion

The genus Abiotrophia most resembles well-known gram positive cocci in chains and pairs such as streptococci and enterococci. Abiotrophia were originally thought to be a relative of the viridans Group Streptococcus but sequencing analysis created a new genus called Abiotrophia which consisted of two species, A. defective and A. adiacens.¹ Abiotrophia is classified as nutritionally variant streptococcus (NVS) and is a part of normal flora in the intestinal tract, oral cavity, and urogenital tract. The organism accounts for up to 6% of streptococcal infective endocarditis and have been associated with native and prosthetic valve infection.² Other types of infections that have been described for Abiotrophia include sepsis, infections of the eye, central nervous system, musculoskeletal, and arthritis.^3-6 It is crucial to recognize A. defectiva for timely and effective treatment, given its propensity to cause severe complications like septic embolization.⁷

Abiotrophia is coined a NVS because this microorganism requires specific nutrients like L-cysteine or pyridoxal for growth on sheep’s blood agar. They can usually grow without supplementation on chocolate agar, brucella agar with 5% horse blood, and in thioglycolate broth. Sometimes sheep’s blood agar supplemented with pyridoxal can also be used. Due to the release of specific nutrients by lysed red blood cells, the satellite test is important for growth and identification. Briefly, the suspected isolate is streaked for confluent growth on an agar that does not support growth for Abiotrophia (such as sheep’s blood agar) and then overlaid with a cross streak of beta-hemolytic Staphylococcus aureus. After incubation, colonies of Abiotrophia can grow near the S. aureus streak, giving rise to the ‘satellite’ phenomenon. However, it should be noted that other fastidious organisms such as Granulicatella and some Haemophilus species can also satellite around the S. aureus streak. Hence, further confirmation is needed for identification. For example, Abiotrophia is non-motile. Common biochemicals reactions to help characterize Abiotrophia genus is PYR (production of pyrrolidonyl arylamidase) positive, LAP (production of leucine aminopeptidase) positive, and no growth in 6.5% NaCl.

Molecular approaches have been proven to be accurate for identification of Abiotrophia . Sequencing of the 16S rRNA gene have shown to be more accurate than phenotypic, biochemical tests. PCR testing for the rpoB, groESL, and also the ribosomal 16S-23S intergenic spacer region (ITS) genes have all shown to be reliable targets for identification.^8-10 The introduction of MALDI-TOF mass spectrometry has facilitated more accurate identification in clinical settings.¹¹

Prompt diagnosis and targeted antimicrobial therapy are essential in managing infections caused by Abiotrophia defectiva, especially in older patients with underlying health conditions. Early detection and appropriate treatment are vital to mitigate potential complications.^6,7 Abiotrophia species exhibit varying antibiotic susceptibility, with increasing resistance to penicillin. Common treatments include penicillin, ceftriaxone, and vancomycin.

References

1.         Kawamura, Y., et al., Transfer of Streptococcus adjacens and Streptococcus defectivus to Abiotrophia gen. nov. as Abiotrophia adiacens comb. nov. and Abiotrophia defectiva comb. nov., respectively. Int J Syst Bacteriol, 1995. 45(4): p. 798-803.

2.         Christensen, J.J. and R.R. Facklam, Granulicatella and Abiotrophia species from human clinical specimens. J Clin Microbiol, 2001. 39(10): p. 3520-3.

3.         Niu, K. and Y. Lin, Abiotrophia defectiva Endocarditis Complicated by Stroke and Spinal Osteomyelitis. Cureus, 2024. 16(3): p. e56904.

4.         Wilhelm, N., et al., First case of multiple discitis and sacroiliitis due to Abiotrophia defectiva. Eur J Clin Microbiol Infect Dis, 2005. 24(1): p. 76-8.

5.         Taylor, C.E. and M.A. Fang, Septic arthritis caused by Abiotrophia defectiva. Arthritis Rheum, 2006. 55(6): p. 976-7.

6.         Yang, M., et al., Abiotrophia defectiva causing infective endocarditis with brain infarction and subarachnoid hemorrhage: a case report. Front Med (Lausanne), 2023. 10: p. 1117474.

7.         Faria, C., et al., Mitral Valve Subacute Endocarditis Caused by Abiotrophia Defectiva: A Case Report. Clin Pract, 2021. 11(1): p. 162-166.

8.         Drancourt, M., et al., rpoB gene sequence-based identification of aerobic Gram-positive cocci of the genera Streptococcus, Enterococcus, Gemella, Abiotrophia, and Granulicatella. J Clin Microbiol, 2004. 42(2): p. 497-504.

9.         Hung, W.C., et al., Use of groESL as a target for identification of Abiotrophia, Granulicatella, and Gemella species. J Clin Microbiol, 2010. 48(10): p. 3532-8.

10.       Tung, S.K., et al., Identification of species of Abiotrophia, Enterococcus, Granulicatella and Streptococcus by sequence analysis of the ribosomal 16S-23S intergenic spacer region. J Med Microbiol, 2007. 56(Pt 4): p. 504-513.

11.       Christensen, J.J., et al., Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis of Gram-positive, catalase-negative cocci not belonging to the Streptococcus or Enterococcus genus and benefits of database extension. J Clin Microbiol, 2012. 50(5): p. 1787-91.

-Maher Ali, MD was born in and raised in Amman, Jordan. He attended Jordan University of Science and Technology, where he received his doctorate degree. After graduation, he completed two years of anatomic pathology residency at King Hussein Cancer Center in Jordan. He then relocated to the United States to start his combined anatomic and clinical pathology (AP/CP) training at The George Washington University. His academic interests include gastrointestinal and breast pathology. Outside of the hospital, he enjoys cooking, hiking, exploring new restaurants, and spending quality time with his family and friends.

-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. She is also a top 5 honoree among the 2023 ASCP 40 Under Forty.

Case History

A 36 year old male with no past medical history presented to an outside hospital with two weeks of worsening headache, fever, and altered mental status. The patient initially presented to his primary care physician with a headache, for which he received an antibiotic injection. Over the next several days, he developed a fever to 39.4^oC, worsening mental status, and bowel incontinence. Once admitted, he experienced an intractable seizure, was subsequently intubated, and transferred to the ICU. CT imaging revealed multiple ring-enhancing lesions throughout the brain, most prominent in the right parietal lobe. Additionally, he was found to have a new diagnosis of HIV (CD4 count 40). He was transferred to our institution for a higher level of care but experienced a rapid progression of disease with deterioration of brainstem reflexes and marked cerebral edema, prompting a brain biopsy.

Laboratory Identification

Frozen and permanent sections of a right temporal biopsy revealed frankly necrotic brain tissue with amoebic cysts and trophozoites, consistent with free-living amoeba (Figure 1 and 2). Cerebrospinal fluid (CSF) cytology was unremarkable. A specific immunohistochemistry (IHC) assay for Acanthamoeba species was performed at the Centers for Disease Control and Prevention (CDC; Atlanta, GA), confirming the diagnosis of granulomatous amoebic encephalitis from Acanthamoeba.

Discussion

Acanthamoeba are free-living amoebae found worldwide in a variety of environmental sites, including soil, water, sewage, swimming pools, contaminated contact lens solution, and heating, ventilating, and air conditioning systems.^1,2 The Acanthamoeba lifecycle consists of two stages: cysts and trophozoites. Although trophozoites represent the infective form, both cysts and trophozoites can enter the body through various means, including the eye, nasal passages, or broken skin.^2,3. There are three distinct diseases that can be caused by Acanthamoeba: Acanthamoeba keratitis, disseminated infection, and Granulomatous Amebic Encephalitis (GAE).¹

GAE is a parenchymal disease that occurs when Acanthamoeba enters the body through the respiratory system or broken skin, spreads hematogenously, and invades the central nervous system.^2,3. Risk factors include immunocompromised status, such as HIV/AIDS, history of organ transplant, uncontrolled diabetes mellitus, liver cirrhosis, and malignancy.^1,3 The clinical course tends to be subacute to chronic, with progressive neurological decompensation over weeks to months and eventual death. Imaging may reveal ring-enhancing lesions, arterial occlusions, infarctions, ventricular shifts, and areas of abnormal enhancement.³ CSF analyses may show a pleocytosis with lymphocyte predominance, increased protein, and decreased glucose; amoebae are typically not identified.^3. While microscopic evidence of double-walled cysts and/or trophozoites demonstrates the presence of free-living amoeba in infected tissues, diagnosis of Acanthamoeba must be confirmed via specific IHC assays or PCR-based molecular techniques.⁴

There is currently no standardized recommended treatment, and no single agent has been shown to be universally effective.^3. Rare successful cases have involved prolonged therapy using combinations of pentamidine isethionate, sulfadiazine, amphotericin D, azithromycin, flucytosine, and fluconazole or itraconazole.^3,5

The patient was treated with miltefosine, fluconazole, pentamidine, flucytosine, and sulfadiazine, sulfamethoxazole/trimethoprim and azithromycin for HIV prophylaxis, and levetiracetam for seizure prophylaxis. Though repeat MRI of the brain/brain stem showed interval improvement of disease burden, he remained unresponsive to verbal, tactile, and painful stimuli, and his overall prognosis remained guarded. The patient was discharged to a long-term acute care facility, but returned to the emergency department one week later with recurrent seizures, hypotension, and fevers. Imaging revealed new areas of restricted diffusion in the right occipital and left parietal lobes. Despite continued treatment, the patient continued to decline and died approximately three months following his initial diagnosis.

References

¹ Centers for Disease Control and Prevention. Parasites – Acanthamoeba – Granulomatous Amebic Encephalitis (GAE); Keratitis. https://www.cdc.gov/parasites/acanthamoeba/index.html. Accessed April 24, 2024.

² Centers for Disease Control and Prevention. DPDx- Free Living Amebic Infections. https://www.cdc.gov/dpdx/freeLivingAmebic/index.html. Accessed April 24, 2024.

³ Siddiqui R, Khan NA. Biology and pathogenesis of Acanthamoeba. Parasit Vectors. 2012 Jan 10;5:6. doi: 10.1186/1756-3305-5-6. PMID: 22229971; PMCID: PMC3284432.

⁴ Haldar SN, Banerjee K, Modak D, Mondal A, Sharma C, Vasireddy T, Karad RK, Patel HB, Majumdar D, Bhattacharjee B, Khurana S, Ghosh T, Guha SK, Saha B. Case Report: A Series of Three Meningoencephalitis Cases Caused by Acanthamoeba spp. from Eastern India. Am J Trop Med Hyg. 2024 Jan 9;110(2):246-249. doi: 10.4269/ajtmh.23-0396. PMID: 38190743; PMCID: PMC10859797.

⁵ Visvesvara GS, Moura H, Schuster FL. Pathogenic and opportunistic free-living amoebae: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, and Sappinia diploidea. FEMS Immunol Med Microbiol. 2007 Jun;50(1):1-26. doi: 10.1111/j.1574-695X.2007.00232.x. Epub 2007 Apr 11. PMID: 17428307.

-Cristine Kahlow, MD, Pathology PGY-1 at UTSW Medical Center

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

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

Case History

A 70 year old female with a medical history of treatment-related acute myeloid leukemia with absolute neutropenia due to chemotherapy presented to our emergency department with a two-day history of fever (Tmax 102°C) with progressively increasing erythema and tenderness reported in her right elbow around a healing surgical incision. Approximately one month prior to presentation the patient underwent surgical excision of her right upper extremity (RUE) cephalic vein for supportive thrombophlebitis as a complication of peripheral IV insertion. Additionally, two weeks prior to presentation the patient was noted to have three small areas (< 1 cm) of wound dehiscence along the surgical incision without overt signs of infection at her outpatient surgical follow-up visit (Figure 1A). These wounds were packed with iodoform gauze and wrapped with sterile kerlix gauze wrap followed by an elastic wrap. The patient was instructed to change the packing and dressing one to two times daily; however, the patient reported non-compliance with these instructions and the packing and dressing had not been changes in the two weeks from the clinic visit until ED presentation.

The patient was admitted to the hospital. Blood cultures were drawn and IV vancomycin, cefepime and metronidazole were initiated for empirical antimicrobial therapy in the setting of neutropenic fever and RUE cellulitis. 18 hours after collection both sets of blood cultures grew a gram positive rod. The infection diseases service was subsequently consulted to determine the significance of the gram positive rods growing in blood culture, if it represented contaminant vs a true pathogen. Imaging of the arm was recommended to further evaluate the extent of the RUE skin & soft tissue infection. CT imaging of the humerus and forearm revealed a thick-walled fluid collection concerning for abscess formation along the operative site involving both the arm and forearm of the patient (Fig. 2). The plastic surgery service was also consulted and noted old iodoform gauze in the areas of wound dehiscence with surrounding erythema (Fig. 1B); however, no purulence was noted from these areas. The erythema surrounding the wound continued to progress (Fig. 1C) during the hospitalization and the patient remained febrile despite the broad-spectrum antibiotic regiment. Given the patient treatment failure by medical management, the patient was taken for surgical washout and debridement of the wound. Deep cultures were obtained during the surgery. Both the tissue culture and blood cultures would grow Corynebacterium jeikeium. The patient developed a maculopapular cutaneous rash several days into the admission which was deemed to be an antibiotic-induced rash. The antimicrobial regiment was narrowed down to daptomycin due to the concern that vancomycin and/or cefepime may be responsible for the drug rash. The cellulitis and abscess resolved following surgical debridement and treatment with vancomycin/daptomycin; however, the patient elected to pursue comfort care status due to her underlying malignancy. She was transitioned to inpatient hospice where she would die shortly afterwards due to progression of her AML.

Laboratory

Corynebacterium species are non-motile, facultatively anaerobic, club-shaped gram positive rods that have a characteristic picket fence appearance when stained due to snapping division. These organisms are commonly encountered in clinical samples; however, given their low virulence in general and prevalence as common skin flora they are often considered contaminants or colonizers. The clinical significance of diphtheria toxin (DT)-producing strains has been known for decades. Non-DT-producing  Corynebacterium species were historically considered as non-virulent but in recent years several non-DT-producing Corynebacterium species have been noted to be pathogenic. The wide spread availability of MALDI-TOF MS in most clinical microbiology labs has allowed for rapid and accurate species level identification of Corynebacterium allowing for identification of these pathogenic species in clinical settings. Some Corynebacterium species are highly lipophilic due to the presence of fatty acids, such as mycolic acid, in their cell wall. Isolation of lipophilic Corynebacterium species on standard culture media can be challenging but can be accomplished utilizing blood agar, due to the presence of lipid-containing red cell membranes, with at least 48 hours of incubation. For optimal recovery of lipophilic strains agars supplemented with Tween 80 produce the best recovery rates.

As a lipophilic species, C. jeikeium can be difficult to identify and propagate. It does not grow well on chocolate agar (Fig. 3A & 3C). On blood agar it tends to have scant or dusky growth at 24 hours (Fig. 3B) and appear as translucent, pinpoint colonies at 48 hours (Fig. 3D). Identification using MALDI-TOF can be challenging before 48 hours due to its poor growth on standard media. For comparison, C. striatum, a non-lipophilic Corynebacterium spp. grows relatively well on both blood and chocolate agar. Several Corynebacterium species, including C. jeikeium and C. striatum, are known to be multidrug resistant, especially towards beta-lactam antibiotics.

Discussion

Corynebacterium jeikeium was initially identified in 1976 and classified by CDC as group JK diptheroids. It is considered part of normal skin flora like most other Corynebacterium spp. In immunosuppressed individuals, especially in persons with hematolymphoid malignancies, invasive disease including dissemination infections have been reported. Infections can also be seen in hardware-associated and prosthetic joint-related infections. Due to its predilection for biofilm formation, C. jeikeium can be difficult to treat in these cases. Cases of infective endocarditis have been reported as well. Like many Corynebacterium species, C. jeikeium is often multidrug resistant. Vancomycin is the first line agent for treatment of infections due to C. jeikeium. Linezolid and daptomycin are other alternative treatment options. When isolating C. jeikeium from blood sources especially in immunocompromised patients like in our case, physicians and other health-care providers must thoroughly assess the total clinical picture before dismissing it as a contaminant.

References

1. Bernard K., Identification of Gram-Positive Bacteria (2023) in AL Leber & CAB Burnham (Eds.) Clinical Microbiology Procedures Handbook (5^th ed.) doi:10.1128/9781683670438.CMPH.ch3.18

2. Gupta R, Popli T, Ranchal P, Khosla J, Aronow WS, Frishman WH, El Khoury MY. Corynebacterium Jeikeium Endocarditis: A Review of the Literature. Cardiol Rev. 2021 Sep-Oct 01;29(5):259-262. doi: 10.1097/CRD.0000000000000355. PMID: 32976125.

3. Mattos-Guaraldi AL, Sanchez Dos Santos L, & Vieira VV, Coryneform Gram-Positive Rods (2023) in Carroll et al. (Eds.) Manual of Clinical Microbiology (15^th ed.) doi:10.1128/9781683670438.MCM.ch28

4. Moore Pardo SM, Patel RH, Ramsakal A, Greene J. Disseminated Corynebacterium jeikeium Infection in Cancer Patients. Cureus. 2020 Jun 22;12(6):e8764. doi: 10.7759/cureus.8764. PMID: 32714702; PMCID: PMC7377673.

5. Murray BE, Karchmer AW, Moellering RC Jr. Diphtheroid prosthetic valve endocarditis. A study of clinical features and infecting organisms. Am J Med. 1980 Dec;69(6):838-48. doi: 10.1016/s0002-9343(80)80009-x. PMID: 7446550.

6. Ozdemir S, Aydogan O, Koksal Cakirlar F. Biofilm Formation and Antimicrobial Susceptibility of Non-Diphtheria Corynebacterium Strains Isolated from Blood Cultures: First Report from Turkey. Medeni Med J. 2021;36(2):123-129. doi: 10.5222/MMJ.2021.60252. Epub 2021 Jun 18. PMID: 34239764; PMCID: PMC8226407.

7. Tauch A, Kaiser O, Hain T, Goesmann A, Weisshaar B, Albersmeier A, Bekel T, Bischoff N, Brune I, Chakraborty T, Kalinowski J, Meyer F, Rupp O, Schneiker S, Viehoever P, Pühler A. Complete genome sequence and analysis of the multiresistant nosocomial pathogen Corynebacterium jeikeium K411, a lipid-requiring bacterium of the human skin flora. J Bacteriol. 2005 Jul;187(13):4671-82. doi: 10.1128/JB.187.13.4671-4682.2005. PMID: 15968079; PMCID: PMC1151758.

-Arooj Devi MD is a second-year pathology (AP/CP) resident at Brody School of Medicine at East Carolina University. She is interested in breast and gynecological pathology.

-John E. Markantonis DO D(ABMM) is the head of microbiology at ECU Health Medical Center and an assistant professor at Brody School of Medicine at East Carolina University. 

A 28 year old woman underwent an elective myomectomy for menorrhagia caused by fibroids. Postoperatively, she developed fevers. A CT scan of the abdomen and pelvis showed a complex pelvic fluid collection measuring 5.4 by 4.4 by 7.0 cm. Drainage was attempted by Interventional Radiology without success. She then underwent an exploratory laparotomy with drainage of the collection, evacuation of a hematoma, and removal of an IUD. The abscess fluid was sent to the Microbiology lab for culture.

The Gram stain of the fluid revealed 1+PMN with no organisms. After 48 hours of incubation, there was few to moderate growth of pinpoint clear colonies on blood agar, with characteristic miniscule appearance of a central area of dense growth and peripherally less dense (Figure 1). Upon closeup reviewing of the colonies revealed a “fried egg” appearance (Fig 2).

There was no growth on MacConkey plate. Final identification by MALDI-TOF demonstrated Mycoplasma hominis.

Discussion

Mycoplasma hominis is a facultative anaerobe of the Mycoplasma genus, which is among the smallest free-living organisms known. They are fastidious and differentiated from other bacteria by their small size and absence of cell walls. Infections with M. hominis predominately involve the urogenital tract of females, causing pelvic inflammatory disease, bacterial vaginosis, and postpartum fever.¹ Extragenital manifestations of M. hominis infections are rare but include extragenital abscesses, CNS infections in neonates, and septic arthritis.² Reports of Mycoplasma hominis infections vary based on demographics (country, age, and number of sexual partners) but have been isolated in 4 to 30% of urogenital infections in females. ^1,3

The lack of cell walls confers both diagnostic and clinical challenges. Mycoplasmas is not seen on the routine Gram stain smears. Instead of a cell wall, they have a triple-layered membrane composed of sterol.³ When able to grow in culture, colonies are small and have a ‘fried egg’ appearance on agar.⁴ Of the Mycoplasma species, M. hominis is the least fastidious and the most common Mycoplasma isolated on conventional culture agar media. ^4-6 Non-traditional culture-based diagnosis is often made via molecular testing. ⁷ It is often detected in coinfections with Trichomonas vaginalis and thought to be a symbiotic relationship. ^8-9

Since M. hominis is also found to be colonized in the genital tract of normal healthy individuals, it can be challenging to establish the clinical and diagnostic significance of M. hominis. With evolving molecular diagnostic assays targeting STI agents, it has been a controversial topic for assay manufacturers or labs that develop their own in-house assays whether there is a clinical utility for M. hominis PCR as part of STI multiplex PCR panels.

In contrast to Mycoplasma pneumoniae, M. hominis has intrinsic resistance to macrolides. ¹⁰ Preferred treatment regimens for M hominis infections include tetracyclines, clindamycin and fluoroquinolones. ¹¹ However, resistance to members of these antibiotic classes has been reported and differs based on country of origin. ¹¹ The absence of a cell wall explains the inherent resistance of Mycoplasma species to the beta-lactam antibiotic class. Its isolation in coinfections, particularly Trichomonas has been controversial in its contribution to emerging Trichomonas metronidazole resistance. ¹²

References

1. Verteramo, R., Pastella, A., Calzolari, E., et al. An epidemiological survey of Mycoplasma hominis and Ureaplasma urealyiticum in gynaecological outpatients, Rome, Italy. Epidemiology & Infection,2013, 141(12), 2650-2657
2. Zheng X, Olson DA, Tully JG, Watson HL, Cassell Gh, Gustafson DR, Svien KA, Smith TF: Isolation of Mycoplasma hominis from a brain abscess. J Clin Microbiol. 1997, 35: 992-994.
3. Thomas Prescott Atkinson, Mitchell F. Balish, Ken B. Waites, Epidemiology, clinical manifestations, pathogenesis and laboratory detection of Mycoplasma pneumoniae infections, FEMS Microbiology Reviews, Volume 32, Issue 6, November 2008, Pages 956–973
4. Razin S. Mycoplasmas. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 37.
5. Meloni GA, Bertoloni G, Busolo F, Conventi L. Colony morphology, ultrastructure and morphogenesis in Mycoplasma hominis, Acholeplasma laidlawii and Ureaplasma urealyticum. J Gen Microbiol. 1980;116(2):435-443.
6. Christiansen G, Jensen LT, Boesen T, Emmersen J, Ladefoged SA, Schiotz LK, Birkelund S: Molecular biology of Mycoplasma. Wien Klin Wochenschr. 1997, 109: 557-561
7. Baczynska, A., Svenstrup, H.F., Fedder, J. et al. Development of real-time PCR for detection of Mycoplasma hominis. BMC Microbiol 4, 35 (2004).
8. Tine RC, Dia L, Sylla K, Sow D, Lelo S, Ndour CT. Trichomonas vaginalis and Mycoplasma infections among women with vaginal discharge at Fann teaching hospital in Senegal. Trop Parasitol. 2019 Jan-Jun;9(1):45-53.
9. Vancini, R.G., Benchimol, M. Entry and intracellular location of Mycoplasma hominis in Trichomonas vaginalis . Arch Microbiol 189, 7–18 (2008).
10. Pereyre S, Gonzalez P, De Barbeyrac B, Darnige A, Renaudin H, Charron A, Raherison S, Bébéar C, Bébéar CM. Mutations in 23S rRNA account for intrinsic resistance to macrolides in Mycoplasma hominis and Mycoplasma fermentans and for acquired resistance to macrolides in M. hominis. Antimicrob Agents Chemother. 2002 Oct;46(10):3142-50.
11. Krausse R, Schubert S. In-vitro activities of tetracyclines, macrolides, fluoroquinolones and clindamycin against Mycoplasma hominis and Ureaplasma ssp. isolated in Germany over 20 years. Clin Microbiol Infect. 2010;16(11):1649-1655.
12. Dessì, D., Margarita, V., Cocco, A., Marongiu, A., Fiori, P., & Rappelli, P. (2019). Trichomonas vaginalis and Mycoplasma hominis: New tales of two old friends. Parasitology, 146(9), 1150-1155. doi:10.1017/S0031182018002135

-Dr. Alex Shaffer is a first-year ID fellow (2023-2025) at Montefiore Einstein. Alex is interested in the diagnostic stewardship projects and has been actively involved in various activities with ID and micro lab faculty.

-Phyu Thwe, Ph.D, D(ABMM), MLS(ASCP)^CM is Associate Director of Infectious Disease Testing Laboratory at Montefiore Medical Center, Bronx, NY. She completed her medical and public health microbiology fellowship in University of Texas Medical Branch (UTMB), Galveston, TX. Her interests includes appropriate test utilization, diagnostic stewardship, development of molecular infectious disease testing, and extrapulmonary tuberculosis.

Case History

A 53 year old male presented to the emergency department with a one-day history of malaise, bilateral flank pain and decreased urine output. His past medical history was notable for decompensated cirrhosis due to alcohol use disorder complicated by esophageal varices and gastric ulcers, peritoneal ascites, several recent episodes of upper GI bleeding, monoclonal gammopathy of unknown significance, and remote prostate cancer status post prostatectomy. Oral history indicated the patient was actively drinking shortly before presentation and had recently consumed oysters on the half shell, broiled shrimp, and crab meat. While being seen in the emergency department, the patient quickly progressed to septic shock.     

Admission laboratory studies demonstrated severe metabolic acidosis, pancytopenia, and acute renal failure. Initial CT abdomen/pelvis demonstrated cirrhotic liver changes, varices, distal esophageal wall thickening, bilateral perinephric fat stranding extending into the pelvis and perivesical fat. The patient was admitted to the MICU for intubation/mechanical ventilation and administration of multiple pressors, as well as empiric meropenem, vancomycin and micafungin given concern for septic shock.Initial blood cultures drawn in the ED flagged positive with gram negative rods (both aerobic and anaerobic bottles) less than 24 hours after collection.

Laboratory Identification

Blood cultures were processed for culture workup and molecular identification. No identification was able to be established using a commercial multiplex PCR-based molecular panel on the positive blood culture broth. Subsequent growth of the organism on MacConkey agar 18 hours later revealed non-lactose fermenting colonies (Image 1A). The organism was oxidase-negative, positive for catalase and indole (Image 1B) and exhibited hydrogen sulfide production when inoculated on triple sugar iron (TSI) media. The organism was definitively identified as Edwardsiella tarda by MALDI-TOF MS and was broadly susceptible to all antibiotic tested including beta-lactams and fluoroquinolones.

Discussion

Edwardsiella tarda is an infrequently isolated member of the Enterobacterales which is most often associated with gastroenteritis. Reports of additional presentations include peritonitis, intra-abdominal abscess, and wound infections are increasing.¹ Bacteremia is rare, and can lead to cholangitis, cholecystitis, and liver abscess via hematogenous spread.     Patients of advanced age (>65 years) and those with hepatobiliary diseases including liver cirrhosis and alcohol abuse and iron storage disorders are at increased risk for extraintestinal disease.^.2 Colonization of the gastrointestinal tract is believed to be a precipitating even leading to bacteremia. Cases of gastroenteritis are usually self-limiting, although ciprofloxacin or trimethoprim/sulfamethoxazole can be utilized in cases of prolonged duration.⁵ Prompt and accurate diagnosis of E. tarda bacteremia is important as this presentation is associated with an elevated mortality rate,⁴ particularly among those with liver disease.     

As members of the Enterobacterales, Edwardsiella sp. share common biochemical features of other members of the order including being catalase-positive and oxidase-negative. Oxidase negativity aids in the distinction between E. tarda and Aeromonas/Plesiomonas, both species which are also associated with aquatic environments and cause gastrointestinal infections.⁵ E. tarda is non-lactose fermenting and reduces sulfur containing amino acids to hydrogen sulfide. This can result in confusion with members of the genus Salmonella⁴ as colonies exhibit similar appearances on medias including Hektoen Enteric agar and Xylose-lysine deoxycholate agar formulated for recovery and presumptive identification of Salmonella from gastrointestinal specimens. Importantly, E. tarda is also indole-positive which will biochemically differentiate these two genera.

E. tarda is associated with freshwater or brackish aquatic environments. This organism is a well-known pathogen in aquaculture industries causing serious infections in fish and significant economic loss. As such, vaccines and prophylactic antibiotics are utilized to prevent E. tarda infection among aquatic animals, and diagnostic approaches including specialized multiplex PCRs are available. Human infection is often associated with consumption of contaminated fish and seafood, and diagnosis is almost exclusively dependent on microbiological culture by contrast. Clinical multiplex molecular panels for both gastrointestinal and bloodstream infections lack the ability to detect E. tarda. In this patient’s case, recent consumption of seafood serves as the most likely event leading to E. tarda gastrointestinal colonization and is consistent with previously reported cases of E. tarda bacteremia.³ The patient was treated with meropenem eventually narrowed to piperacillin/tazobactam and blood cultures cleared. He was weaned off pressors and extubated.     Unfortunately, the patient decompensated over the course of three weeks due to worsening shock and acidosis. He was moved to comfort care and expired soon thereafter.

References

1. Hirai et. al. 2015.  Edwardsiella tarda bacteremia. A rare but fatal water- and foodborne infection: Review of the literature and clinical cases from a single centre. Can. J. Infect. Dis. Med. Microbiol. 26(6): 313-318.
2. Hasegawa M., and Sanmoto, Y.  2024. Recurrent cholangitis and bacteremia due to Edwardsiella tarda: a case report.     Oxford Med. Case Rep. Volume 2024, Issue 1, January 2024, omad148, https://doi.org/10.1093/omcr/omad148
3. An et. al.     2023.     Case Report: Disseminated Edwardsiella tarda infection in an immunocompromised patient.     Front. Cell. Infect. Microbiol. 20 November 2023.
4. Janda, J.M, and Abbott, S.L. 1993. Infections Associated with the genus Edwardsiella: the Role of Edwardsiella tarda in human disease. Clin. Infect. Dis. Oct;17(4):742-8.
5. Janda, J.M and Abbott, S.L. 1999. Unusual Food-Borne Pathogens: Listeria monocytogenes, Aeromonas, Plesiomonas, and Edwardsiella species. Clin. Lab. Med. 19(3):553-582.

-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.

-Francesca Lee, MD, is an associate professor in the Departments of Pathology and Internal Medicine (Infectious Diseases) at UT Southwestern Medical Center

Case description

A 72 year-old male with severe aortic stenosis resulting in heart failure underwent aortic valve replacement and became febrile with mild shortness of breath on post-operative day one.  Additional pertinent medical history includes end-stage renal disease secondary to diabetic nephropathy with kidney transplantation seven months prior which was complicated by delayed graft function requiring hemodialysis and immunosuppression. The patient’s presentation was suspicious for either a post-operative fever or a complication of hemodialysis. Physical examination did not suggest infection at either the surgical site or his arteriovenous fistula. A chest CT revealed a moderate, loculated left pleural effusion with left lower lobe atelectasis and nodular consolidations in the right lung, raising concern for pneumonia. A bronchial biopsy was obtained, and histopathological evaluation was consistent with an abscess, but no organisms were visualized. Culture from a left bronchial brushing was also obtained at the same time. Two days later, the patient developed a progressively enlarging forehead lesion (Image 1).

Laboratory Identification

Microscopic evaluation of the biopsy of the lesion for culture revealed branching, beaded gram positive filamentous bacteria which stained positive using a modified acid-fast stain, suggestive of Nocardia species. This was corroborated by the bronchial culture, where similar modified acid-fast organisms were visualized and grew chalky white colonies identified as Nocardia sp. by MALDI-TOF MS (Image 2). The isolate was referred to a reference laboratory for definitive speciation and antimicrobial susceptibility testing, and the patient was started empirically on ceftriaxone and trimethoprim/sulfamethoxazole.  Speciation and susceptibility testing subsequently identified the organism as Nocardia brasiliensis with susceptibility to both ceftriaxone and trimethoprim/sulfamethoxazole, and treatment was thereafter continued for 40 days.

Discussion

Nocardia is a saprophytic bacterium that is commonly found in soil worldwide. Gram staining of Nocardia sp. usually reveals delicate, weakly or erratically staining, beaded, branching Gram-positive rods. There are currently 130 known species of Nocardia,¹ of which more than 50 have been reported to have clinical relevance.² Disease due to Nocardia is known to occur due to inhalation of the organism from the environment or traumatic inoculation. It is thought to be an opportunistic pathogen most commonly affecting immunocompromised patients³ like the patient in this case.

Manifestations of nocardiosis vary widely depending on the species responsible, with most common presentations being lymphocutaneous, pulmonary, or disseminated disease. Here, we present a patient who displayed both lymphocutaneous and pulmonary forms of Nocardia infection. While prognosis is poorer in immunocompromised patients, with appropriate treatment, recovery rate is high in both lymphocutaneous and pulmonary forms, unlike in disseminated disease, where studies have found mortality rates between 44% to 85%.⁴

Diagnosis of Nocardia infections is mainly performed through either direct visualization of the organism or through culture. The modified acid-fast stain is commonly performed but has limited sensitivity and should generally be performed in conjunction with the Gram stain.⁵ Recovery in culture is most reliable from respiratory and/or tissue samples, while blood culture, by contrast, has poorer yield. While some strains, as in this patient, appear within several days in culture, some strains may take up to 2 to 3 weeks to detect⁵ necessitating extended incubation and consultation with the clinical laboratory.

Treatment requires prolonged courses of antibiotics. Susceptibilities vary by species, making it important to obtain species identification to identify appropriate therapy to guide empiric therapy. Susceptibility testing is performed to allow further tailoring of antibiotic regimens. In this case, the patient was treated with a combination of trimethoprim/sulfamethoxazole and ceftriaxone, both of which cover the majority of clinically relevant Nocardia species, until susceptibility testing revealed that the empiric treatment provided adequate coverage for the Nocardia brasiliensis identified, supporting continuation of the chosen empiric regimen.

References

1. Genus Nocardia. List of Prokaryotic names with Standing in Nomenclature. https://www.bacterio.net/genus/nocardia (2023).
2. Hamdi AM, Fida M, Deml SM, Abu Saleh OM, Wengenack NL. Retrospective Analysis of Antimicrobial Susceptibility Profiles of Nocardia Species from a Tertiary Hospital and Reference Laboratory, 2011 to 2017. Antimicrob Agents Chemother. 2020 Feb 21;64(3):e01868-19. doi:10.1128/AAC.01868-19.
3. McNeil MM, Brown JM. The medically important aerobic actinomycetes: epidemiology and microbiology. Clin Microbiol Rev. 1994 Jul;7(3):357-417. doi:10.1128/CMR.7.3.357.
4. Traxler RM, Bell ME, Lasker B, Headd B, Shieh WJ, McQuiston JR. Updated Review on Nocardia Species: 2006-2021. Clin Microbiol Rev. 2022 Dec 21;35(4):e0002721. doi: 10.1128/cmr.00027-21.
5. Saubolle MA, Sussland D. Nocardiosis: review of clinical and laboratory experience. J Clin Microbiol. 2003 Oct;41(10):4497-501. doi: 10.1128/JCM.41.10.4497-4501.2003.

-Albert Budhipramono, MD, PhD, is currently a PGY1 Clinical Pathology Resident at the University of Texas Southwestern Medical Center in Dallas, Texas.

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

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

What is the test?

In the 1920s, the Greek physician Georgios Papanikolaou developed a method of cervical cancer screening, now reliably used and colloquially known as a “Pap smear”. During a Pap smear, a healthcare provider swabs cells from the cervix for further analysis by the lab​¹​, including cytopathologic examination. Regular utility of Pap smears in women aged 21 to 65 has decreased the incidence and mortality of cervical cancer by at least 80% in the United States alone​²​. 95% of cervical cancer cases worldwide are caused by persistent human papilloma virus (HPV) infection of the cervix​³​. However, other sexually transmitted infections (STIs) and common non-STIs with associated morphological changes can also be identified on Pap smears. 

Bacteria

Bacterial vaginosis (BV) is the most common cause of abnormal discharge in young women, characteristically with a “fishy” ammonia-like odor. Although not strictly considered an STI, this infection is associated with severe rare complications including pelvic inflammatory disease (PID), infertility and premature labor. The vagina typically have an abundance of Lactobacillus spp., which is a gram positive bacilli (Figure 1). However, if the normal vaginal flora is disrupted with high abundance of gram negative bacteria such as Gardnerella vaginalis or Mobiluncus, then BV may occur. On pap smears, ‘clue cells’ which are squamous epithelial cells with coccobacilli, appear as dark purple staining and cloudy appearance suggest BV. In cervicovaginal samples typically found in women using intrauterine device usage, clumps of filamentous bacteria suggestive of Actinomyces species may also be visible. For cases pertaining to bacterial etiologies, staining of the rods or filaments usually warrant suspicion of infection. Other the other hand although Chlamydia trachomatis is the most common bacterial STI in the United States, with up to 4 million new cases every year, diagnosis on pap smears is not the definitive diagnosis since the cytopathology is non-specific. The interpretations include visualization of inflammatory exudates and inflammatory cells. Oftentimes, further testing is needed. 

Parasite

Trichomonas vaginalis is the most prevalent non-viral STI in the United States, strongly associated with an increased risk of HIV infection among women​⁴​. Majority of the patients may exhibit symptoms such as burning, itching and vaginal discharge. T. vaginalis is a parasitic flagellated protozoan although in some cases, a flagella may be found. Typically, it is of a pear-shape and staining of a nucleus may be visualized (Figure 2).

Fungus

Yeast infections (vulvovaginal candidiasis) due to Candida infection occur in 75% of women at some point in their lives. Classic clinical presentation includes itching, erythema, and thick white “cottage-cheese” discharge. There is also an increased association with HIV infection, diabetes, and any cause of immunosuppression (e.g. transplant, chemotherapy, steroids). Morphologically, budding yeast forms (conidia, small and oval measuring 3-6 µm) and pseudohyphae (long filamentous spores) are found. The combination of pseudohyphae and yeast forms are referred to as “sticks and stones” and frequently, squamous cells lined up along the pseudohyphae are found referred to as having “shish kebab” appearance (Figure 3)⁵. Note that no true septation is found.

On pap smears, sometimes there could be superficial mucosal infections that may appear associated with enlarged hyperchromatic nuclei with halos, which can be confused with low grade squamous intraepithelial lesions.

Viruses

HPV with its associated risk of cervical cancer remains the most crucial microorganism to detect on Pap smears. Given HPV’s association to cancer development, it is crucial to examine the samples for cervical lesions and their associated pathologies. HPVs in the low-risk category typically is associated with low-grade squamous intraepithelial lesions and HPVs in the high-risk category is associated with high-grade squamous intraepithelial lesions and invasive squamous cell carcinoma.  Other viral etiologies seen in PAP smears include Herpes simplex virus (HSV) and cytomegalovirus (CMV). Infections with HSV can be asymptomatic but the most common symptom is the development of vesiculopustular or small ulcerative lesions on the genitalia. The classic cytopathological findings of HSV infection are the 3 “Ms”: multinucleation, nuclear molding, and margination of chromatin. CMV infections are rare, usually asymptomatic and can be transient. On the pap smears, the infected cells may appear with intranuclear inclusions surrounded by a halo.

Conclusions

While Pap smears are routinely performed on women and can provide a presumptive diagnosis, current developments in molecular technologies (e.g. Nucleic acid amplification tests (NAATs)) is transforming the field. There are several FDA-approved molecular platforms available for clinical diagnostic labs to test for HPV and can even genotype the strain to determine risk levels. Crucially, the remaining sample from liquid-based Pap tests​⁶​ can also be submitted for both NAAT testing and HPV-DNA testing, facilitating a quicker turnaround time and obviating the need for additional patient sampling.  Most recently, a PCR-based test is now on the market (Cepheid, Sunnyvale, CA) that can diagnose all three etiologies, BV, Candidiasis, and Trichomoniasis, within 60 minutes from a single specimen.  In summary, accurate examination of the Pap smear often incidentally provides the first step in the diagnosis and further work-up of all these infectious diseases.

References 

​​1. Pap Smear: MedlinePlus Medical Test [Internet]. [cited 2024 Feb 4]. Available from: https://medlineplus.gov/lab-tests/pap-smear/&nbsp;

​2. PDQ® Screening and Prevention Editorial Board. Cervical Cancer Screening (PDQ®): Health Professional Version. National Cancer Institute [Internet]. 2022 [cited 2024 Feb 4];1–26. Available from: https://www.cancer.gov/types/cervical/hp/cervical-screening-pdq&nbsp;

​3. Lei J, Ploner A, Elfström KM, Wang J, Roth A, Fang F, et al. HPV Vaccination and the Risk of Invasive Cervical Cancer. New England Journal of Medicine. 2020 Oct 1;383(14):1340–8.  

​4. Davis A, Dasgupta A, Goddard-Eckrich D, El-Bassel N. Trichomonas vaginalis and Human Immunodeficiency Virus Coinfection Among Women Under Community Supervision: A Call for Expanded T. vaginalis Screening. Sex Transm Dis [Internet]. 2016 Sep 15 [cited 2024 Feb 5];43(10):617–22. Available from: https://pubmed.ncbi.nlm.nih.gov/27631355/&nbsp;

​5. Kamal Meherbano M.  The Pap smear in inflammation and repair. Cytojournal [Internet]. 2022 Apr 30; 19:29. Available from: doi: 10.25259/CMAS_03_08_2021

​6. Hawthorne CM, Farber PJ, Bibbo M. Chlamydia/gonorrhea combo and HR HPV DNA testing in liquid-based pap. Diagn Cytopathol [Internet]. 2005 Sep [cited 2024 Feb 5];33(3):177–80. Available from: https://pubmed.ncbi.nlm.nih.gov/16078250/&nbsp;

-Zoon Tariq is a pathology resident at George Washington University. Her interests include surgical pathology and cytopathology.

-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 59-year-old man presented to the Emergency Room with bright red blood per rectum, associated with nausea, vomiting, abdominal cramping, and persistent watery diarrhea. Several days earlier, he had returned from a three-week trip to the Dominican Republic. On physical examination, he was afebrile. His abdomen was soft and not tender or distended.

A stool sample was sent to the Microbiology Lab for PCR testing, and both Vibrio and Vibrio cholera targets were detected. The stool was then plated for culture confirmation. Hemolytic colonies grew on the blood agar plate (Figure 1), and yellow (original medium color green) colonies grew on thiosulfate citrate bile sucrose selective agar due to sucrose fermentation (Figure 2). Gram stain from these colonies showed gram negative, curved, comma-shaped rods (Figure 3) and MALDI-ToF identification revealed Vibrio albensis. The specimen was sent to the Department of Public Health for confirmation, which reported Vibrio cholerae O1 serovar Ogawa with O1 antigen typing. 

Discussion

Vibrio albensis is a gram negative, halophilic bacterium belonging to the Vibrionaceae family. V. albensis is a recently identified species within the Vibrio genus and is believed to be a member of the Vibrio cholera complex. Although primarily considered non-pathogenic, V. albensis has been associated with rare cases of human infections, particularly in individuals with compromised immune systems.¹ While previously reported studies indicated V. albensis as a non-O1, non-O130 serogroup of V. cholerae,²the Department of Public Health confirmed our patient’s isolate as O1 serovar Ogawa by O1 antigen typing. V. albensis is an emerging pathogen with limited information regarding its clinical significance and optimal management. The infections are predominantly associated with contaminated seawater or seafood exposure. The primary transmission mode is through open wounds or ingesting raw or undercooked seafood.³

Clinical presentation of this organism can be similar to V. cholerae, as diffuse watery diarrhea, the hallmark of cholera, or asymptomatic. Other case reports presented bacteremia, septicemia, and urinary tract infections.^4,5

Laboratory diagnosis of Vibrio cholerae albensis infection involves isolating and identifying the bacterium from stool samples. This can be achieved using selective culture media, such as thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which allows for the growth of V. cholerae and its variants.⁶

Antimicrobial resistance is a growing concern in the management of cholera. Studies have reported varying resistance levels to commonly used antibiotics in V. cholerae, including V. cholerae albensis. It is essential to monitor antimicrobial susceptibility patterns to guide appropriate treatment strategies.⁷ Further research is needed to better understand the epidemiology, clinical manifestations, and optimal treatment strategies for V. albensis infections as members of V. cholerae complex are being identified/recognized with more advanced diagnostic tools.

References

1. Baker-Austin C, et al. (2016). Vibrio albensis sp isolated from a mesophilic bacterial culture, abalone (Haliotis spp.), and seawater. International Journal of Systematic and Evolutionary Microbiology, 66(1), 187-192.
2. Ahmed AOE, Ali GA, Hassen SS, Goravey W. Vibrio albensis bacteremia: A case report and systematic review. IDCases. 2022 Jun 30;29:e01551. doi: 10.1016/j.idcr.2022.e01551. PMID: 35845827; PMCID: PMC9283503.

3. Sharma P, et al. (2018). Vibrio albensis: An Emerging Pathogen Causing Necrotizing Fasciitis. Journal of Clinical Microbiology, 56(3), e01454-17.

4. Araj GF, Taleb R, El Beayni NK, Goksu E. Vibrio albensis: An unusual urinary tract infection in a healthy male. J Infect Public Health. 2019 Sep-Oct;12(5):712-713. doi: 10.1016/j.jiph.2019.03.018. Epub 2019 Apr 10. PMID: 30981654.

5. Sack RB, et al. (2004). Cholera. The Lancet, 363(9404), 223–233.

6. Centers for Disease Control and Prevention (CDC). (2021). Laboratory Methods for the Diagnosis of Vibrio cholerae. Retrieved from https://www.cdc.gov/cholera/laboratory.html

7. Ceccarelli, M., et al. “Editorial–Differences and similarities between Severe Acute Respiratory Syndrome (SARS)-CoronaVirus (CoV) and SARS-CoV-2. Would a rose by another name smell as sweet.” European review for medical and pharmacological sciences 24.5 (2020): 2781-2783.

-Eros Qama, MD, is a 2nd year AP/CP pathology resident in the Department of Pathology at Montefiore Medical Center in Bronx, NY

-Phyu Thwe, Ph.D, D(ABMM), MLS(ASCP)^CM is Associate Director of Infectious Disease Testing Laboratory at Montefiore Medical Center, Bronx, NY. She completed her medical and public health microbiology fellowship in University of Texas Medical Branch (UTMB), Galveston, TX. Her interests includes appropriate test utilization, diagnostic stewardship, development of molecular infectious disease testing, and extrapulmonary tuberculosis.

Case History

An 83 year old female with history of type 2 diabetes presented to the emergency room (ER) with two month history of dysuria and diarrhea. Upon admission, Clostridiodes difficile (C. difficile ) GDH/TOX with reflex to nucleic acid amplification test (NAAT), urine culture, and 2 sets of blood culture specimens were collected for testing. C. difficile test was positive for GDH and Toxin A/B production. Urine culture was positive with 50,000 – 99,000 colony forming units (CFU)/mL of Klebsiella pneumoniae. Patient was administered ceftriaxone, metronidazole and vancomycin and discharged three days post admission. On day of discharge, blood cultures were documented as “no growth at 3 days”. The patient visited her primary care physician the day after discharge. Two days later, the primary care office was notified of a corrected report. The patient’s blood culture collected on day of admission was being corrected from no growth to a positive with growth of Fusobacterium nucleatum. The patient was contacted to check on her status and medication compliance was verified. Patient continued to demonstrate symptom improvement during follow up at physician’s office 10 days post discharge.

What happened here?

The blood culture was collected at an acute care hospital. The system microbiology laboratory policy for a first blood culture positive that is read as no organisms seen (NOS) from an acute care hospital laboratory, is to inoculate appropriate media and prepare three smears. Gram stain is to be performed on one of the smears and if organisms are not seen, a stained and unstained smear, along with inoculated media, are to be sent to the core microbiology laboratory (core lab). The bottle is then to be reloaded on the blood culture instrument.

The policy for a second positive alert from the same NOS bottle is to inoculate appropriate media and prepare 3 cytospin smears. Gram stain is to be performed on one of the cyto-smears and if organisms are still not seen, a stained and unstained smear are to be sent to the core lab along with the positive bottle and inoculated media. The core lab is to use the unstained cytospin smear to make and read an acridine orange (AO) stain.

In this patient’s case, the anaerobic blood culture bottle from one set flagged as positive on day 4 of incubation. The gram stain was read as NOS and inoculated media and smears were sent to the core lab. The bottle was reloaded on the instrument and the bottle alerted as positive a second time that day. The cytospin gram stain was read as NOS and inoculated media, smears, and blood culture bottle were sent to the core lab. At the core lab, a new unspun smear was made from the bottle. AO was performed on this smear and read as NOS.

In our laboratory, if a gram stain result is not entered for a blood culture, it will auto verify as a negative blood culture every 24 hours with a final negative result at 5 days. Both blood culture sets on this patient resulted as no growth at 5 days.

At the 48 hour (6 days post-collection) culture read, BAP, CHOC, and MAC demonstrated no growth, but the CDC ANA had growth. The isolate was setup for MALDI identification and resulted as 99.9% Fusobacterium nucleatum.

Per protocol, a review of smears was performed on this discrepant smear-culture. The unstained cyto-smear sent by the acute care laboratory had not been stained with AO and read per protocol. Rather, as was mentioned above a Gram stain was made on a new unspun smear and read as NOS. Reviewing this AO stain during the investigation revealed long thin fluorescing rods with tapered ends (Fig 1)

The cyto-smear that had been sent after the second positive alert, but did not get stained and read was also stained with AO and long thin fluorescing rods with tapered ends were observed (Fig 2).

At this point in the investigation, the unspun gram stain from the 1^st positive alert that was sent over from the acute care hospital laboratory was also reviewed. Knowing that the bottle was positive for F. nucleatum the technologist shared that she eventually saw rare long thin gram-negative rods with pointed ends that matched what was seen in the AO (Fig 3). As part of the investigation, safranin was added for an additional 10 minutes revealing the organism more clearly (Fig 4).

Discussion

Fusobacterium is an obligate anaerobic gram-negative rod that gram stains as a light staining thin rod with pointed ends. Fusobacterium are found in oropharyngeal flora and are commonly seen in oral biofilms. It is a primary pathogen seen in peri-implantitis, root canal infections, dentoalveolar abscesses and spreading odontogenic infections. It can also be a pathogen seen in abscesses in various parts of the body and seen in the blood stream.

Due to the staining characteristics of Fusobacterium species, they often blend into the background of gram stains from positive blood cultures. As a result, the miss in gram stain and delay in culture growth combined with the late detection of Fusobacterium on blood culture instrumentation for these fastidious organisms can result in a false negative report that is only caught after the organism grows from the original NOS bottle.

Safranin is a secondary stain or counterstain, utilized in the Gram stain, that will stain the colorless gram negative bacteria pink or red. Legionella, Brucella melitensis, Legionella and Campylobacter species are all reported to be enhanced with safranin left on for 2 minutes and it is recommended if anaerobes are suspected and not seen to leave on for 3-5 minutes or use basic fuchsin in order to enhance the morphology of these organisms. For this reason, some laboratories routinely use basic fuchsin as the counterstain.

Acridine orange (AO) is a fluorochromatic dye which binds to nucleic acids of microorganisms and human cells. Acridine orange is a fluorochrome stain that binds to the nucleic acid of cells and bacteria.  RNA and single‐stranded DNA will appear orange and double‐stranded DNA found in human cells, with the exception of red blood cells, will appear yellow or yellow‐green, when visualized under UV light. Therefore, bacteria and fungi stain bright orange while epithelial, white blood cells, and background debris will appear pale green to yellow. To name a few, some common applications for AO include routine screening of normally sterile body fluids and rule‐out of Gram‐positive microorganisms versus crystal violet stain precipitate. Of important note, while not able to differentiate the actual organism, it does work for detecting Mycoplasma and Ureaplasma.

Conclusion

Blind subculturing of NOS gram stains from positive blood cultures, longer staining with safranin, and AO stains are beneficial to be added to the micro lab armamentarium. They are especially beneficial when added into the protocol for processing sterile body site specimens in which organisms, that stain lightly and blend in the background, may be suspected. For further review, Special Media or Stains for Fastidious and Infrequently Encountered Organisms can be found in the Clinical Microbiology Procedures Handbook 5^th edition.

References

Kononen E, Nagy E, Conrads G. 2023. Bacteroides, Porphyromonas, Prevotella, Fusobacterium, and Other Anaerobic Gram-Negative Rods. Manual of Clinical Microbiology 13^th edition. ASM Press, Washington, DC.

Tille PM.  Bailey & Scott Diagnostics Microbiology, 14th ed., St. Louise, Mosby, Inc., 2017.

Dallas SD and Harrington A. 2023. 3.2 Staining Procedures. Clinical Microbiology Procedures Handbook 5^th edition. ASM Press, Washington, DC.

-Jennifer Tedrick MLS(ASCP), Technical Specialist

-Maureen Bythrow, M(ASCP), Microbiology Manager

-Frances Valencia-Shelton, PhD, D(ABMM), SM(ASCP)^CM is the Clinical Infectious Diagnostics Director for the Baptist Health System in Jacksonville, FL. She is actively engaged in the Jacksonville Area Microbiology Society and the American Society for Microbiology. Her interests include defining and utilizing clinical best-practice for testing and reporting. She is equally interested in learning with and educating others in the field of clinical microbiology.