1. Taxonomy and characteristics of the Nocardia genus
- Classification system of Nocardia
According to the most recent classification system, Nocardia spp. currently comprise 109 validly named species, of which approximately half are considered clinically relevant1. The taxonomy of Nocardia is continuously evolving, and the application of 16S rRNA gene sequencing, multilocus sequence analysis (MLSA), and whole-genome sequencing (WGS) has significantly refined the classification and identification of this genus1˒10.
The prevalence of specific Nocardia species varies substantially depending on the identification methodology, clinical presentation, and geographic region1. When molecular techniques such as MLSA are applied, Nocardia cyriacigeorgica and Nocardia farcinica emerge as the most prevalent species overall1˒3˒4. Notably, many isolates previously reported as Nocardia asteroides have been reclassified as N. cyriacigeorgica based on molecular characterization1. In contrast, Nocardia brasiliensis is the predominant agent of primary cutaneous nocardiosis and nocardial actinomycetoma, rather than a universally prevalent species across all clinical syndromes1. Other commonly encountered clinically relevant species include Nocardia nova, Nocardia otitidiscaviarum, Nocardia veterana, and members of the Nocardia transvalensis complex1˒3.
Geographic factors and host immune status also significantly influence species distribution. For example, N. brasiliensis is more frequently reported in tropical and subtropical regions, while N. farcinica and N. cyriacigeorgica are commonly identified in temperate climates1˒3. In immunocompromised patients, certain species such as N. farcinica and N. cyriacigeorgica are associated with more severe disseminated infections2˒6.
Accurate species identification is critical for developing effective treatment strategies, as different Nocardia species exhibit distinct antimicrobial susceptibility profiles. For instance, N. farcinica is intrinsically resistant to many β-lactams11, while N. brasiliensis often shows reduced susceptibility to carbapenems3˒4. Thus, precise species identification directly guides appropriate antimicrobial therapy and improves clinical outcomes.
- Biological characteristics of Nocardia
Nocardia spp. are Gram-positive, branched filamentous bacteria with a strong ability to adapt to various niches, enabling them to survive under diverse environmental conditions12. These bacteria typically form filamentous colonies and produce characteristic “chalky” or “wrinkled” colonies on agar media (Figure 1). Nocardia grows slowly, usually requiring several days to weeks of incubation for visible growth. In addition, Nocardia spp. can utilize various carbon sources, demonstrating strong metabolic diversity, which allows survival in a wide range of natural environments13.
- Infectious characteristics of Nocardia
Nocardia infections typically present as pulmonary infections, skin infections, or systemic diseases and are more common in immunocompromised patients2˒14. Clinically, manifestations are diverse and may include cough, fever, and difficulty breathing. Infections can rapidly progress to severe pulmonary disease or sepsis. In some cases, Nocardia infections can also cause brain abscesses or other central nervous system infections, particularly in immunosuppressed patients15. Therefore, early identification and prompt treatment are critical for improving patient prognosis.
- Epidemiological features of Nocardia infections
Epidemiological studies show significant geographic variation in Nocardia infections. The global incidence is relatively low but increases markedly in high-risk populations, such as HIV-infected individuals and organ transplant recipients. For example, the incidence of Nocardia infections in kidney transplant recipients is approximately 0.4%–1.3%, whereas in lung transplant recipients it can be as high as 9%16˒17.
Susceptibility analyses across different populations indicate that patients receiving long-term immunosuppressive therapy are more prone to Nocardia infections, which are closely associated with compromised immune function2. Additionally, environmental exposure factors, such as soil contact and dust inhalation, are considered to increase the risk of Nocardia infection, particularly among individuals living or working in high-risk environments18.
2. Advances in molecular identification techniques for Nocardia spp.
- 16S rRNA gene sequencing
Due to the complex and time-consuming process of cultivating and isolating Nocardia, the introduction of PCR technology has significantly improved diagnostic speed and accuracy. Laurent et al. demonstrated that amplification of the 16S rRNA gene using specific primers could rapidly identify Nocardia from cultured isolates, laying the groundwork for its application in clinical microbiology19. Subsequently, direct detection of Nocardia from clinical specimens using 16S rRNA PCR has been reported, although its sensitivity remains variable and requires prior clinical suspicion20˒21.
Direct comparative studies have shown that Nocardia-specific qPCR targeting the 16S rRNA gene provides higher sensitivity and shorter turnaround time than conventional culture in clinical specimens, including bronchoalveolar lavage fluid, sputum, pus, and tissue samples. Couble et al. reported a sensitivity of 88% and specificity of 98% for direct Nocardia qPCR, with results available within hours, whereas culture may require days to weeks of incubation22. Ding et al. further confirmed that 16S rRNA gene PCR can be directly applied to clinical specimens for Nocardia detection, allowing rapid identification even in culture-negative cases and effectively compensating for the limitations of traditional culture21.
This method effectively confirms nocardial infection but, as a targeted approach, requires prior clinical suspicion. Despite its high accuracy for genus-level identification, the high conservation of the 16S rRNA gene poses challenges for distinguishing closely related species, such as those within the Nocardia asteroides complex10,21. This limitation reflects reduced species-level resolution rather than low overall accuracy. Therefore, sequencing results often require cross-validation with other methods, such as multilocus sequence analysis (MLSA) or whole-genome sequencing (WGS), to achieve definitive species-level resolution23˒24.
- Multilocus sequence analysis (MLSA)
MLSA is a molecular technique with significant application value in bacterial species identification and typing23. For complex genera such as Nocardia, MLSA enables more accurate species identification by analyzing sequence information from multiple conserved genes. Studies have demonstrated that MLSA can effectively differentiate among various Nocardia species, particularly those that are morphologically and physiologically similar25. For example, one study identified 14 different Nocardia species by sequencing four genes (e.g., gyrB, hsp65, secA1, and 16S rRNA), highlighting the efficiency and accuracy of MLSA25. Furthermore, MLSA can reveal genetic diversity and support epidemiological investigations. It also facilitates tracking of strain transmission and variation, providing important data for infection monitoring and control.
However, MLSA is labor-intensive, costly, and lacks universal cutoff thresholds, limiting its routine clinical application. As noted by Traxler et al., MLSA is not well-suited for routine use due to these constraints1. Therefore, the primary role of MLSA in current practice is limited to research settings, including epidemiological investigations, strain typing, and taxonomic studies. For routine clinical identification, MALDI-TOF MS is preferred because of its speed, low cost, and ease of use. When higher resolution is required—such as for definitive species identification, resistance gene detection, or phylogenetic analysis—whole-genome sequencing (WGS) offers superior capabilities. Recent studies using WGS have not only distinguished known Nocardia species with high resolution but have also enabled the identification of potential novel species, surpassing the capabilities of MLSA alone24. WGS has therefore emerged as a more powerful alternative, offering higher resolution and improved analytical capability9.
- Application and challenges of MALDI-TOF MS
Matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS) has become increasingly widespread in microbiology, particularly for rapid pathogen identification, demonstrating significant clinical value26. This technology analyzes microbial protein spectra to provide accurate species- or complex-level identification within a short time. For example, studies have reported a 97.3% identification accuracy for Nocardia species in clinical samples in Japan27.
Despite its strong performance, challenges remain. Species-level identification accuracy can vary depending on the database version and the species tested23. Updated databases have been shown to improve the proportion of isolates achieving reliable genus-level identification (score >1.7) from 77.6% to 94.7%27, highlighting the importance of regular database updates. Species-level identification typically requires higher score thresholds (e.g., >2.0).
MALDI-TOF MS offers rapid identification (typically <10 min per isolate after colony growth), low cost per test ($1–5 per sample), and minimal technical complexity, making it well-suited for frontline clinical microbiology laboratories. However, limitations include the high initial instrument cost and the need for comprehensive, regularly updated spectral databases to maintain species-level accuracy.
- Breakthroughs in whole-genome sequencing (WGS)
Whole-genome sequencing (WGS) is increasingly demonstrating significant advantages in microbial identification by providing comprehensive genomic information and enabling phylogenetic analysis, thereby offering insights into bacterial evolutionary relationships28. Studies have shown that WGS enables detailed characterization of the genetic diversity and population structure of Nocardia24.
The feasibility of implementing WGS in clinical laboratories is improving. For example, WGS can identify emerging variants and provide data for antimicrobial resistance monitoring29. However, despite its high accuracy and comprehensive output, widespread adoption remains limited by high costs and the complexity of data analysis.
Although WGS is becoming a reference method, barriers such as cost, operational complexity, and infrastructure requirements persist. In most clinical laboratories, WGS is impractical for routine diagnostics due to costs ($100–300 per isolate), turnaround times (3–7 days), the need for advanced bioinformatics infrastructure, and specialized expertise. Currently, its use is largely restricted to reference laboratories and outbreak investigations.
- Metagenomic next-generation sequencing (mNGS)
Metagenomic next-generation sequencing (mNGS) has introduced new possibilities for detecting Nocardia30. Traditional culture methods often face challenges due to the slow growth and specific environmental requirements of Nocardia, leading to delayed diagnosis. In this context, mNGS has emerged as a valuable alternative.
Several case reports and small-scale studies have demonstrated the potential of mNGS for Nocardia detection31. For instance, mNGS has been used to diagnose cutaneous Nocardia infection, rapidly identifying the pathogen in immunocompetent patients32. Similarly, in ear, nose, and throat infections, mNGS identified N. farcinica, enabling targeted treatment33. In patients with chronic obstructive pulmonary disease, mNGS analysis of respiratory samples successfully identified N. otitidiscaviarum, improving diagnostic timeliness and accuracy34. Additionally, mNGS can detect mixed infections, as demonstrated in cases where both N. farcinica and other pathogens were identified30.
However, these findings are largely based on case reports or small cohorts and require validation in larger, prospective studies. Therefore, conclusions regarding the diagnostic performance of mNGS for Nocardia should be interpreted with caution, and the technology remains a complementary tool rather than a first-line diagnostic method.
Despite its promise, mNGS has several important limitations. High cost ($500–1,500 per sample) and limited availability restrict its use to specialized centers or complex cases7˒31. The lack of standardized protocols contributes to variability in performance7˒30. Interpretation is challenging due to difficulty distinguishing true infection from colonization or contamination, particularly in respiratory samples30˒31˒35. Moreover, mNGS does not provide antimicrobial susceptibility data, which are critical for guiding therapy1˒4. Finally, the clinical significance of low-abundance sequences remains uncertain, and standardized reporting thresholds are lacking7˒31.
Overall, mNGS represents a valuable adjunctive diagnostic tool, particularly in complex or critically ill patients when conventional methods are inconclusive. However, it should not be considered a universal diagnostic solution, and results must be interpreted alongside clinical, radiographic, and microbiological findings.
- Exploration of emerging molecular diagnostic methods
With advances in molecular biology, emerging diagnostic approaches are being explored for Nocardia detection. CRISPR-based detection technologies enable rapid identification of specific gene sequences with high sensitivity36. In addition, machine learning and artificial intelligence (AI) approaches have introduced new possibilities for rapid screening of Nocardia37.
However, these findings are derived from small-scale, proof-of-concept studies and should be interpreted with caution. At present, none of these approaches has been clinically validated for routine Nocardia detection. CRISPR-based assays may eventually evolve into rapid, low-cost point-of-care tools, but this will require extensive clinical validation. Similarly, AI-assisted image analysis has shown preliminary potential for screening but lacks standardized protocols and prospective evaluation. Both approaches remain in early development and are not yet recommended for clinical use. Future studies should focus on multicenter validation, standardization, and demonstration of clinical utility before these methods can be adopted into routine practice.
To facilitate comparison, the key performance characteristics, practical constraints, and recommended clinical roles of these methods are summarized in Table 1.
3. Antimicrobial susceptibility testing (AST) of Nocardia spp.
- Resistance mechanisms of Nocardia
The resistance mechanisms of Nocardia species primarily include gene mutations, enzyme production, and biofilm formation1. Many Nocardia species, particularly N. farcinica and N. cyriacigeorgica, have demonstrated resistance to multiple antibiotics38. For example, resistance to sulfonamides in Nocardia spp. is associated with mutations in the dihydrofolate reductase gene, resulting in decreased susceptibility to trimethoprim–sulfamethoxazole (TMP-SMX)39,40,41.
Additionally, Nocardia species can produce β-lactamases and other enzymes that degrade antibiotics, leading to resistance to penicillins and cephalosporins42. These resistance mechanisms complicate the treatment of Nocardia infections, particularly in high-risk populations, where the emergence of resistant strains further exacerbates therapeutic challenges. The major resistance mechanisms identified in Nocardia species are summarized in Table 2, including specific resistance genes, mutation types, and quantitative susceptibility data.
- Traditional antimicrobial susceptibility testing
Traditional antimicrobial susceptibility testing methods primarily include microdilution and agar diffusion techniques. The microdilution method is considered the most reliable for determining the antimicrobial susceptibility of Nocardia, providing accurate minimum inhibitory concentration (MIC) values43.
Despite being the reference method, broth microdilution for Nocardia is subject to significant inter- and intra-laboratory variability. Factors such as inoculum preparation, choice of culture medium, incubation time, and difficulties in endpoint interpretation due to trailing growth or poor turbidity can affect reproducibility35˒43. These technical challenges are particularly relevant for slow-growing and heterogeneous organisms such as Nocardia and underscore the importance of standardized protocols1.
In one study, 146 Nocardia strains were tested using microdilution, showing high susceptibility to linezolid, amikacin, and TMP-SMX, with rates of 100%, 96%, and 94%, respectively4˒44. However, these data originate from a single geographic region (Japan) and should not be generalized without caution. Antimicrobial resistance profiles of Nocardia vary substantially by geographic region, influenced by local antibiotic usage patterns and circulating species1˒3. The agar diffusion method is also widely used for susceptibility testing in Nocardia. However, its accuracy is influenced by factors such as culture medium, inoculum density, and the slow growth rate of Nocardia, which can result in poorly defined inhibition zones1˒43. Unlike broth microdilution, agar diffusion has not been standardized for Nocardia by the Clinical and Laboratory Standards Institute (CLSI) and should be considered a screening method rather than a reference technique.
Although traditional methods remain effective in clinical practice, increasing diversity and resistance among Nocardia species have led to growing interest in integrating molecular detection methods to provide a more comprehensive assessment. The microdilution method remains the gold standard for antimicrobial susceptibility testing; however, it is dependent on bacterial growth rate, which can be problematic for slow-growing species such as Nocardia. The disk diffusion method is more convenient but less sensitive for such organisms. Molecular techniques, including PCR-based detection of resistance genes, provide rapid alternatives but remain supplementary and cannot replace phenotypic testing.
- Application of molecular detection methods
With advances in molecular biology, molecular detection methods are increasingly used to complement traditional phenotypic approaches for identification, while their role in antimicrobial susceptibility testing remains an active area of research, primarily focused on detecting specific resistance genes35.
For example, 16S rRNA gene sequencing and MLSA can differentiate Nocardia species and provide insight into associated resistance genes21. Whole-genome sequencing (WGS) enables comprehensive genomic analysis, offering deeper insights into genetic composition and resistance mechanisms24˒28. Some studies have identified various β-lactamase genes in N. farcinica11, which are closely associated with antibiotic resistance38.
Metagenomic next-generation sequencing (mNGS) also demonstrates advantages in identifying resistant strains. In some cases, mNGS can identify Nocardia species and provide resistance-related information, supporting antibiotic selection and reducing inappropriate treatment due to misdiagnosis30.
However, the current understanding of genotype–phenotype correlations in Nocardia remains incomplete, and many resistance determinants remain poorly characterized. Therefore, molecular approaches cannot replace phenotypic susceptibility testing at present. They serve as adjunct tools to support the interpretation of culture-based results and may guide targeted therapy when well-characterized resistance genes are detected. Clinical decisions should continue to rely primarily on phenotypic AST results, particularly given geographic variability in resistance patterns and the lack of standardized interpretive criteria for molecular methods.
4. Clinical challenges in Nocardia identification and antimicrobial susceptibility testing
Nocardia species are opportunistic pathogens that can cause severe infections, particularly in immunocompromised patients1˒46. Although these infections are relatively rare, their high morbidity and mortality present significant challenges for clinical diagnosis and treatment. Accurate diagnosis of nocardiosis begins prior to laboratory testing, with pre-analytical factors playing a critical role in the success of both culture-based and molecular methods. Specimen quality directly influences detection rates; deep respiratory samples such as bronchoalveolar lavage (BAL), induced sputum, or biopsy specimens yield higher recovery rates than expectorated sputum. Transport conditions are equally important, as Nocardia species are fastidious and may be overgrown by commensal flora if specimens are delayed or improperly stored. Prolonged transport times (>2 h) without appropriate media can compromise viability and reduce culture yield45.
Decontamination procedures used for respiratory specimens (e.g., N-acetyl-L-cysteine–NaOH) may inadvertently reduce Nocardia recovery due to its slow growth and susceptibility to chemical agents. Therefore, laboratories should consider selective media (e.g., buffered charcoal yeast extract agar) and extended incubation periods (up to 2–4 weeks) to improve recovery46. A major clinical challenge is distinguishing colonization from true infection. Nocardia may be isolated from respiratory samples in patients with chronic lung disease (e.g., bronchiectasis, chronic obstructive pulmonary disease) without evidence of invasive disease. In such cases, detection may represent colonization rather than infection. This distinction is critical, as unnecessary treatment may increase toxicity and resistance, whereas failure to treat true infection can result in disseminated disease with high mortality. Clinical judgment, supported by radiographic findings, histopathology, and systemic symptoms, is essential2.
Host factors significantly influence both susceptibility and outcomes. The type and degree of immunosuppression are key determinants. Among solid organ transplant recipients, incidence varies by organ type: lung transplant recipients have the highest risk (up to 9%), followed by heart and liver recipients, whereas kidney transplant recipients have lower but still significant incidence (0.4%–1.3%)16˒17. These differences reflect variations in immunosuppressive regimens and environmental exposure. The use of T-cell-depleting agents and high-dose corticosteroids further increases risk, particularly within the first year post-transplant1.
Mass spectrometry-based technologies, such as MALDI-TOF MS, have demonstrated high accuracy in Nocardia identification, although limitations remain26˒27. Species-level identification accuracy depends heavily on the comprehensiveness and currency of the reference database, and performance varies among different database versions. These factors, rather than species-specific identification failures, represent the primary challenges for routine clinical laboratories23.
Regarding antimicrobial susceptibility testing, significant variability exists among Nocardia species. While many strains remain susceptible to linezolid and amikacin, resistance to other antibiotics varies by species and may influence treatment outcomes3. Therefore, susceptibility testing remains crucial for guiding therapy, as discussed in Section 3.