A Case Report of a Forehead Abscess Caused by Nocardia concava in a Patient with AIDS

Summary

Here, we present a protocol for Nocardia infections in immunocompromised patients that integrates pus culture and MALDI-TOF MS. This combined approach enables rapid, accurate species identification, facilitates targeted antimicrobial therapy, and ultimately improves clinical outcomes in this high-risk patient population.

Abstract

Nocardia species are obligate aerobic actinomycetes. Human nocardiosis typically occurs in immunocompromised hosts and most commonly manifests as primary suppurative pulmonary infection. Hematogenous dissemination can lead to extrapulmonary involvement, with the central nervous system being the most frequent secondary site, followed by skin and subcutaneous soft tissues; pericardium, lymph nodes, and joints are affected only rarely. A 50-year-old HIV-positive man was admitted with a 1-month history of oral blood blisters and ecchymoses of the extremities. A right forehead abscess that developed during hospitalization yielded pus that was identified as Nocardia concava by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and confirmed by 16S rRNA gene sequencing. Initial management included antiretroviral therapy, systemic glucocorticoids, and intravenous immunoglobulin; subsequent targeted antimicrobial therapy with linezolid plus trimethoprim-sulfamethoxazole, instituted upon microbiological confirmation, resulted in clinical improvement and discharge. To our knowledge, this is the first case report of forehead abscess infection caused by N. concava in an acquired immunodeficiency syndrome (AIDS) patient. Human infections due to this organism remain extremely rare. Moreover, the infection site in this case was not the commonly affected lung or central nervous system, typically involved in nocardiosis, which resulted in atypical clinical manifestations prone to missed diagnosis or misdiagnosis. In this case, precise etiological identification allowed for early definitive diagnosis and favorable clinical outcomes. This case underscores the importance of increased vigilance for rare Nocardia infections in severely immunocompromised patients. For unexplained skin and soft tissue abscesses, early etiological examination is critical, and targeted antimicrobial therapy based on drug susceptibility can significantly improve prognosis.

Introduction

Nocardia species are aerobic actinomycetes widely distributed in the natural environment, commonly inhabiting soil and decaying organic matter. Although ubiquitously present in nature, they are typically absent from the human body and function as opportunistic pathogens. Most infections occur via inhalation¹. Nocardiosis predominantly affects immunocompromised individuals, particularly those with impaired cell-mediated immunity, such as patients with malignancies, diabetes mellitus, HIV/AIDS, autoimmune diseases, solid organ transplant recipients, and individuals undergoing prolonged corticosteroid therapy². Nevertheless, infections can also occur in individuals without apparent immunodeficiency³. Nocardia primarily invades via the respiratory tract, causing pulmonary disease, and can subsequently disseminate hematogenously to cause extrapulmonary organ involvement. The brain is the most common site of dissemination, followed by the skin and subcutaneous soft tissues; less commonly, it may involve the pericardium, lymph nodes, and joints. Clinically, Nocardia can cause acute or chronic, localized or disseminated suppurative granulomatous inflammation involving the lungs, skin, and central nervous system⁴.

To date, human infections caused by N. concava have been rarely reported. Herein, we present the first documented case of a forehead abscess in an HIV-positive patient attributed to N. concava, offering valuable insights for the diagnosis and management of nocardiosis.

Case Presentation:
A 50-year-old male patient was admitted to our hospital with a one-month history of oral blood blisters and purpuric lesions on the extremities. He had been diagnosed with HIV infection over ten years prior, but had not adhered consistently to antiretroviral therapy. Approximately one month before admission, he developed oral hematomas and limb purpura without an identifiable trigger and was initially treated at a local hospital with Biktarvy (bictegravir/emtricitabine/tenofovir alafenamide) for antiretroviral therapy. Based on further investigations, immune thrombocytopenia (ITP) was suspected, and he received pulse therapy with methylprednisolone and intravenous immunoglobulin (IVIG), along with gastric protection and calcium supplementation.

Due to persistent thrombocytopenia, the patient was referred to our institution for further management. On admission, his body temperature was 37.9 °C; he was alert, oriented, and in fair general condition. No icterus was observed in the skin or sclera. Widespread petechiae and ecchymoses were noted across the body, and a palpable mass was present on the right frontal region. Non-contrast chest CT revealed interstitial changes in the lower lobes bilaterally and paraseptal emphysema in the upper lobes. Abdominal ultrasound showed no significant abnormalities.

Laboratory tests performed the following day revealed a platelet count of 12 × 10⁹/L(83–303 × 10⁹/L), procalcitonin level of 0.11 ng/mL (<0.50 ng/mL), and high-sensitivity C-reactive protein (CRP) of 1.11 mg/L (<6.00 mg/L). The absolute count of helper/inducer T lymphocytes (CD3+, CD4+) was 15 cells/µL(550–1440 cells/µL). Quantitative Polymerase Chain Reaction(PCR) assays detected Epstein-Barr virus (EBV) DNA at 1.21 × 10⁴ IU/mL (0–200 IU/mL) and human cytomegalovirus (HCMV) DNA at 4.53 × 10⁵ IU/mL (0–200 IU/mL), both positive; other routine blood tests were unremarkable.

Given the confirmed HCMV viremia, the patient was initiated on combination antiviral therapy with ganciclovir and foscarnet, while continuing Biktarvy for HIV suppression. Pulse-dose methylprednisolone and IVIG were maintained for ITP. Due to persistent low-grade fever, rituximab was administered on hospital day 6, and voriconazole was started on day 7 as prophylaxis against oropharyngeal candidiasis.

On admission, physical examination revealed a mass over the right frontal region (Figure 1). On hospital day 6, purulent material was aspirated from this lesion for microbiological evaluation. Direct Gram staining of the pus demonstrated abundant branching, filamentous Gram-positive bacilli (Figure 2). A portion of the specimen was inoculated onto Columbia blood agar plates and incubated at 35 °C in a 5–10% CO₂-enriched atmosphere. After 24 h of incubation, white, raised, dry, wrinkled, granular, non-hemolytic colonies were observed. By day 5 of incubation, the colonies developed a pale yellow pigmentation (Figure 3). Modified acid-fast staining of the isolate yielded partially acid-fast and partially non–acid-fast organisms (Figure 4).

MALDI-TOF MS using the Autobio MS2000 system identified the organism as N. concava (Figure 5). This identification was subsequently confirmed by 16S rRNA gene sequencing, which also classified the isolate as N. concava. Antimicrobial susceptibility testing was performed, and the results are summarized in Table 1. The susceptibility results, interpreted according to the CLSI M24 guidelines for Nocardia spp., indicated susceptibility to all tested agents.

On hospital day 8, following the laboratory report of abundant Gram-positive bacilli, voriconazole was discontinued, and combination antimicrobial therapy with linezolid and trimethoprim–sulfamethoxazole was initiated. The patient's body temperature gradually normalized, and clinical improvement was observed. On hospital day 18, the patient was discharged in improved condition, with prescriptions for oral linezolid and methylprednisolone to continue outpatient treatment.

Diagnosis, Assessment, and Plan:
The patient has AIDS with a low absolute CD4+ T-helper/inducer cell count, predisposing to opportunistic infections. Detectable CMV and EBV DNA indicate possible active cytomegalovirus and Epstein-Barr virus replication. Physical examination revealed a right-forehead abscess and scattered petechiae/purpura. Pus aspirated from the forehead abscess yielded colonies identified as N. concava by MALDI-TOF MS.

Chest CT showed bilateral lower-lobe interstitial changes; follow-up is required to exclude pulmonary nocardiosis or HIV/CMV-related interstitial lung disease. The abscess was incised and drained, thrombocytopenia was managed, and the patient was discharged on oral linezolid plus trimethoprim–sulfamethoxazole for nocardiosis.

Post-discharge monitoring includes weekly CBC and hepatic/renal profiles to watch for myelosuppression and immune reconstitution inflammatory syndrome; immediate re-hospitalisation is required if fever, new nodules, bleeding, or neurological symptoms occur.

Protocol

The study protocols were approved by the Medical Ethics Committee of the First Affiliated Hospital of the College of Medicine, Zhejiang University. (Reference Number: IIT20240874A).

1. Patient assessment and initial evaluation

1. A comprehensive medical history was obtained.
   1. One month prior to presentation, the patient developed oral blood blisters and ecchymoses of the extremities without any identifiable precipitating factors.
   2. The patient subsequently sought care at a local hospital, where Biktarvy was initiated as antiretroviral therapy, and thrombopoietic agents were administered; nevertheless, no hematologic improvement was achieved.
2. On physical examination, the patient was noted to be in a low-grade febrile state. Scattered petechiae and ecchymoses were observed over the entire body surface, and a well-circumscribed mass was present in the right frontal region.
3. Laboratory Investigations
   1. Complete blood count (platelet enumeration): Whole blood (3 mL) was collected into an ethylenediaminetetraacetic acid (EDTA)-containing tube, gently inverted 8–10 times, and analyzed within 4 h of phlebotomy.
   2. Serum C-reactive protein and procalcitonin assays: Blood (2–3 mL) was drawn into a serum separator tube, allowed to clot for 20–30 min, centrifuged at 1,500–2,000 × g for 10 min, and analyzed on the day of collection.
   3. Flow-cytometric enumeration of lymphocyte subsets (CD3/4/8/16/19/45/56): Whole blood (3 mL) was collected into an EDTA tube, gently inverted 8–10 times to ensure adequate anticoagulation, and analyzed within 24 h of collection.
   4. Quantitative polymerase chain reaction (PCR) for Epstein–Barr virus and cytomegalovirus DNA: Whole blood (3 mL) was drawn into an EDTA tube, gently inverted 8–10 times, maintained at 2–8 °C, and transported within 4 h of collection. Plasma was separated by centrifugation within 2–4 h post-phlebotomy and stored at 4 °C pending analysis.

2. Diagnostic procedures

1. Ultrasound-guided aspiration of right forehead abscess:
   1. Following standard skin disinfection and sterile draping, local infiltration anesthesia was administered with 2% lidocaine. Under real-time ultrasonographic guidance, a 20-mL syringe with an attached needle was advanced into the forehead abscess, and 4 mL of purulent fluid was aspirated.
   2. The patient remained hemodynamically stable and reported no discomfort throughout the procedure. No immediate complications were observed in the post-procedural period.
2. The purulent aspirate was forwarded to the microbiology laboratory for immediate Gram staining and subsequent culture in 5–10% CO₂ at 35 °C.
   1. For Gram staining, first, take a clean glass slide to prepare a bacterial smear, fix it over a flame, then apply crystal violet stain for primary staining for 1 min, followed by a water rinse.
   2. Next, add iodine solution as a mordant for 1 min, and rinse with water. Then decolorize with 95% ethanol for approximately 20–30 s until the runoff becomes colorless, and immediately rinse with water to stop decolorization.
   3. Finally, counterstain with safranin solution for 30 s to 1 min, rinse with water, blot dry, and observe under a microscope.
3. A loopful of colonies from the blood agar plate was selected and subjected to modified acid-fast staining.
   1. For modified acid-fast staining, first, take a clean glass slide to prepare a bacterial smear, fix it over a flame, then cover the smear with carbol fuchsin stain and heat until steam rises without boiling, maintaining for 3–5 min, followed by water rinse.
   2. Then, perform weak decolorization with 1% sulfuric acid aqueous solution for 30 s to 1 min until the smear appears light pink, and rinse with water.
   3. Finally, counterstain with methylene blue solution for 30 s to 1 min, rinse with water, blot dry, and observe under a microscope.
4. Identification was performed using an automated mass spectrometry microbial identification system, and the isolate was identified as N. concava.
   1. Sample preparation
      1. Target plate preparation: A single pure colony was picked with a bamboo stick and evenly smeared onto the target spot of the MALDI target plate.
      2. Formic acid addition: After the smear was air-dried, 1 µL of formic acid was added to each target spot for cell lysis.
      3. Matrix addition: After the spot was completely air-dried, 1 µL of matrix solution (α-cyano-4-hydroxycinnamic acid, HCCA) was immediately overlaid, and the plate was allowed to air-dry again.
   2. Power on and set up the software.
      1. Turn on the instrument power, vacuum pump, and computer, and wait until the vacuum reaches the working status. Launch the workstation software and log in.
      2. Confirm normal communication between the software and the instrument, and verify that the vacuum, temperature, and laser status are all normal.
   3. Enter information in the software.
      1. Enter the target plate management module. Select the target plate type, create a new batch, and enter sample information, including sample ID, type, source, and remarks.
      2. Save the target plate information and select the corresponding identification database for comparison.
   4. Operate the instrument and acquire data.
      1. Place the dried target plate into the instrument chamber and close the chamber door. Select Run Target Plate in the software and confirm the target plate ID and position.
         NOTE: The software automatically completes laser excitation, ion flight, signal acquisition, and spectrum generation. Real-time monitoring of the acquisition progress and spectrum quality is available.
   5. Data analysis and identification
      1. Spectrum processing: The software automatically performs smoothing, baseline correction, and peak extraction on the raw spectrum.
      2. Database comparison: The processed characteristic peak spectrum is compared with the standard spectrum database of known bacteria built into the instrument.
      3. Result interpretation: The software provides identification results and scores based on comparison similarity. Generally, a score ≥ 9.0 indicates reliable identification to the species level; a score between 6.0 and 8.9 indicates identification to the genus level; and a score below 6.0 is considered unreliable.
      4. Reporting and data management: Raw spectra, identification results, and logs are automatically saved. Data can be exported, analyzed statistically, and interfaced with the LIS.
   6. Shutdown procedure
      1. After completing all tasks, exit the operation interface. Enter standby mode in the software, wait for the instrument to safely depressurize, close the software and computer, and finally turn off the power to the instrument and the vacuum pump.
5. Antimicrobial susceptibility testing
   1. Pick colonies and grind them with a grinding rod or glass beads in sterile water to prepare a homogeneous suspension, adjust to 0.5 McFarland turbidity standard; since Nocardia tends to form clumps, thorough grinding is required to ensure uniform suspension.
   2. Then add the bacterial suspension to each well of the microdilution plate containing cation-adjusted Mueller-Hinton broth (CAMHB), inoculate 10 µL per well to achieve a final inoculum of approximately 1 × 10⁵–5 × 10⁵ CFU/mL, simultaneously set up a growth control well without antibiotics, and a negative control well.
   3. Incubate the microplate at 35 ±± 2°C in ambient air for 3–5 days, keeping the plate lid sealed during incubation to prevent desiccation.
   4. After incubation, observe bacterial growth in each well with the naked eye, and record the lowest drug concentration that completely inhibits growth as the minimum inhibitory concentration (MIC).

3. Antibiotics therapy

1. Immediately upon admission, combined antiviral and immunosuppressive therapy was initiated: ganciclovir plus foscarnet were administered together with continued Biktarvy for HIV suppression, while pulse therapy with methylprednisolone and intravenous immunoglobulin was concomitantly started.
2. Day 6: Recurrent low-grade fever prompted the addition of rituximab to the therapeutic regimen.
3. Day 7: To preempt possible invasive fungal infection, empirical voriconazole was initiated.
4. Day 8: Microscopy revealed abundant Gram-positive bacilli; voriconazole was discontinued, and antimicrobial therapy was promptly switched to linezolid plus trimethoprim-sulfamethoxazole. Thereafter, defervescence ensued, and the patient's clinical status progressively improved. He was discharged on hospital day 18 in stable condition.

Results

A 50-year-old HIV-positive male patient presented with generalized petechiae and ecchymoses, and oral herpes. After admission, he received empirical antiviral and thrombopoietic therapies. Physical examination revealed a right-forehead mass. Ultrasound-guided aspiration (Figure 1) was performed to obtain pus for laboratory examination. Gram staining results are shown in Figure 2, and acid-fast staining results are shown in Figure 4....

Discussion

Nocardia is an aerobic, Gram-positive bacterium classified within the phylum Actinobacteria, class Actinobacteria, subclass Actinobacteridae, order Actinomycetales, suborder Corynebacterineae, family Nocardiaceae, and genus Nocardia¹⁵. Over 100 species have been identified, with the most common pathogens in humans and animals being N.asteroides, N. brasiliensis, N. farcinica, and N. cyriacigeorgica

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5–10% CO2 Incubator	Thermo Fisher Scientific	20172416507
Acid Fast Stain	Baso	20170094
Autof MS Series	Autobio Diagnostics	https://en.autobio.com.cn/Product/productDetail/fid/63/cid/2/id/145.html
Autof MS2000	Autobio Diagnostics	20182400196
Formic acid	Autobio Diagnostics	20140009
Gram Stain	BioMérieux	20171067
α-cyano-4-hydroxycinnamic acid	Autobio Diagnostics	20140009