Rapid Identification of Cryptococcus Organisms Easily Possible with Stimulated Raman Histology on a Pulmonary Tiny Biopsy Specimen: A Case Report ()
1. Introduction
Cryptococcus neoformans is a globally significant fungal pathogen and a leading cause of opportunistic infections, particularly in immunocompromised populations. This environmental yeast is commonly found in soil, bird excreta (notably from pigeons), and decaying wood, with human infection typically occurring via inhalation of aerosolized spores or yeast cells. While primary pulmonary infections may be asymptomatic or cause mild respiratory symptoms, C. neoformans has a marked ability to disseminate hematogenously, often leading to life-threatening complications such as cryptococcal meningitis, especially in individuals with advanced HIV/AIDS, solid organ transplant recipients, or those undergoing immunosuppressive therapy [1] [2]. Globally, cryptococcal meningitis is estimated to cause over 220,000 cases annually, with a high mortality rate, particularly in resource-limited settings [3].
Despite advances in antifungal treatments, the significant morbidity and mortality associated with cryptococcosis highlight the need for improved diagnostic, prevention, and treatment strategies. Stimulated Raman Histology (SRH) is a form of histology that uses Stimulating Raman Scattering (SRS) to detect molecular vibrations in tissue representing CH2 bonds (in lipids) and CH3 bonds (in DNA/Proteins). The NIO laser imaging system applies a pseudo color mimicking traditional H&E, allowing for intra-procedural viewing of freshly scanned tissue. This case report aims to demonstrate the utility of SRH in the rapid diagnosis of pulmonary cryptococcosis, potentially transforming diagnostic workflows.
2. Case Report
A 44-year-old man with a history of smoking and alcohol-use presented to the hospital with a cough and right sided chest pain. His past medical history was otherwise negative for, and he denied history of, immunosuppressive conditions. He was HIV-negative and had no previous history of malignancy or chemotherapy. CT imaging showed a 3.3 cm spiculated right lower lung mass with a differential diagnosis of infection vs. neoplastic mass lesion. The patient was evaluated by the pulmonology team at Hoag Hospital Newport Beach and underwent an ION robotic-assisted bronchoscopy for biopsy. Hoag Hospital is a two-hospital system with a main 500 bed hospital and satellite 100 bed hospital with several large supporting outpatient centers in and around Orange County, CA. Our facility is evaluating a novel imaging known as Stimulated Raman Histology (SRH) in conjunction with standard of care Hematoxylin and Eosin (H&E) for histologic examination methods. During examination, and documented below, the biopsy revealed a Cryptococcus fungal infection which eventually spread to various parts of the body and required a 6-week hospitalization with anti-fungal therapy under intensive care. SRH was able to identify a cryptococcal infection rapidly and accurately with diagnostic morphologic findings equivalent to the traditional H&E examination histologic standard of care. SRH was performed intraoperatively, taking approximately 90 seconds for acquisition, and provided rapid, accurate diagnostic findings equivalent to traditional H&E examination. Microbiological cultures were performed based on SRH findings, correlating with H&E results.
3. Materials and Methods (SRH)
The NIO laser imaging system by Invenio Imaging was used intraoperatively. Using Laser Raman Scattering the images in Figure 1(a) and Figure 1(b) were obtained in under 5 minutes during the procedure. For comparison, after biopsy completion, the sample was sent to pathology from which Figure 2(a) and Figure 2(b) were obtained from standard H&E-stained specimens. In both preparations, the characteristic polysaccharide capsules (the basis for cryptococcal antigen serologic tests) are easily identified.
(a) (b)
Figure 1. (a) is a Stimulated Raman Histology image obtained from the NIO laser imaging system during bronchoscopy of lung mass, (a clinically suspected metastasis). (b) is a Stimulated Raman Histology image obtained from the NIO laser imaging system during bronchoscopy of lung mass, (a clinically suspected metastasis).
4. Discussion
The rapid pathological assessment of small tissue specimens from mass lesions often centers on excluding malignant and benign neoplasms for tissue triage. Pulmonary specimens, however, uniquely require prioritizing the exclusion of infectious etiologies, particularly in granulomatous diseases. Granulomas can mimic radiologically features of malignancy, displaying necrosis and rapid size changes that complicate initial diagnostic impressions [1] [3]. While molecular testing and culture remain the gold standards for microbial identification, histological examination occasionally reveals fungal organisms if present in sufficient numbers or if their morphology is distinctive [2].
(a) (b)
Figure 2. (a) is a Hematoxylin and Eosin generated image obtained from the pathology department at Hoag Hospital in Newport Beach. (b) is a Hematoxylin and Eosin generated image obtained from the pathology department at Hoag Hospital in Newport Beach.
The expanding arsenal of imaging and biopsy technologies, such as endobronchial ultrasound (EBUS)-directed biopsies and supradimensional fine needle biopsies, has improved access to pulmonary tissue [4] [5]. However, the challenge persists in optimizing the triage of these often-limited specimens for both morphological and microbiological testing. Emerging technologies, such as Stimulated Raman Histology (SRH), provide a promising solution. First validated for rapid, label-free, high-resolution imaging of CNS lesions [6], SRH offers the advantage of preserving tissue integrity without compromising subsequent histological evaluation. Unlike traditional frozen sections that consume and alter tissue during processing, SRH enables non-destructive assessment, making it especially valuable for small specimens [7].
In this case report, we demonstrate the utility of SRH in diagnosing a Cryptococcus pulmonary infection presenting as a mass lesion. The ability of SRH to provide rapid, label-free morphological insights highlights its potential to transform tissue triage workflows. Importantly, SRH can inform whether additional sterile passes are necessary for microbiological testing in suspected infectious cases. Because SRH slides are not sterile, a separate sterile tissue sample is required for culture to ensure accurate microbiological diagnosis without the risk of contamination [8]. However, scanning an SRH slide that suggests fungal or bacterial presence provides immediate feedback to the proceduralist, allowing for targeted specimen collection and limiting the need for repeat procedures.
In large community and academic centers, interventional radiology (IR) and interventional pulmonology (IP) collect initial biopsy specimens, which occasionally may need to be repeated when sampling infectious etiologies. Incorporating SRH into this workflow could streamline diagnosis by enabling rapid intra-procedural morphological assessment [9]. For example, if an SRH scan reveals fungal elements, proceduralists can immediately prioritize collecting additional samples for microbiological testing rather than sending initial samples to fixative or pathology without this critical context. This triage approach minimizes unnecessary delays, reduces repeat procedures, and benefits both patients and healthcare systems.
SRH offers an affordable, portable, and rapid diagnostic tool that does not require specialized cytotechnologists, making it suitable for resource-limited settings. Incorporating SRH into diagnostic workflows can reduce procedure times, minimize repeat biopsies, and improve patient outcomes. Limitations include initial setup costs and the need for training, but its ease of use by existing OR staff mitigates these challenges.
In resource-limited settings where cryptococcosis is prevalent, SRH can facilitate timely diagnosis, reducing morbidity and mortality. The rapid and non-destructive nature of SRH supports its integration into various clinical settings, enhancing diagnostic accuracy and efficiency.
Furthermore, SRH can support diagnostic decision-making in non-infectious cases by confirming malignancy or other neoplastic processes, potentially avoiding unnecessary microbiological testing and preserving specimen integrity [10]. This dual role rapidly identifying infectious or non-infectious pathology enhances the efficiency and accuracy of tissue triage. This non-consumptive system could lead to fewer biopsy passes, resulting in rapid procedure turnaround time with potentially less patient morbidity.
This case highlights an exciting application of SRH beyond its established role in CNS lesions, with implications for rapid, non-destructive pulmonary specimen assessment.
Acknowledgements
The authors would like to express their gratitude to the healthcare professionals at Hoag Memorial Hospital Presbyterian in Newport Beach, CA, for their dedication to patient care and their commitment to advancing medical knowledge. Additionally, we acknowledge the collaborative efforts of the clinical and laboratory teams whose contributions made this innovative diagnostic approach possible. This work is a testament to the importance of teamwork and the pursuit of excellence in healthcare.
Funding
No specific funding was received for this case report. The authors conducted this work as part of their routine clinical practice and commitment to advancing patient care.
Ethical Considerations
This case report adheres to all relevant ethical guidelines and standards. Informed consent was obtained from the patient for the use of medical information and images in this report. All patient data have been de-identified to protect confidentiality.