First report of Nocardia wallacei infection in an immunocompetent patient in Zhejiang province

Abstract Nocardiosis is an infectious disease caused by Nocardia spp., mainly affecting immunocompromised hosts. Nocardia infection is not common; especially Nocardia wallacei infection is even rarer. The patient, female, 61 years old, farmer, has been working in the field for a long time and has normal immune function. Her main clinical manifestation was persistent back pain. Chest-enhanced computed tomography showed pulmonary inflammation. Rare pathogen Nocardia wallacei was detected in alveolar lavage fluid using matrix-assisted laser destructive ionization time-of-flight mass spectrometry. She received treatment with linezolid and was discharged after her condition improved.


Introduction
In recent years, with the increase of HIV infection, solid organ transplantation, hematopoietic stem cell transplantation, autoimmune disease patients treated with corticosteroids, hematological malignancies, and lung structural and functional damage, Nocardiosis has begun to appear frequently in the public health [1].Nocardiosis is a rare bacterial infection caused by Nocardia that usually affects individuals with low immunity and has a high incidence rate and mortality [2,3].Nocardiosis mainly manifests as fever, cough, sputum, chest pain, and other symptoms, which has no characteristic clinical manifestations compared with other infections and make it challenging to diagnose in clinical practice [4].Nocardia is a group of aerobic actinomycetes, mostly saprophytic and mainly distributed in soil.It is an opportunistic pathogenic bacterium that primarily invades the body through the respiratory tract or wounds, causing purulent infections.In severe cases, it can spread to multiple organs through the bloodstream [2,4,5].Currently, over 90 species of Nocardia have been described, of which approximately 30 can cause human diseases [4,6].Nocardia cyriacigeorgica and Nocardia farcinica are considered the main pathogenic species worldwide, while in China, Nocardia asteroides is commonly found [7].Nocardia wallacei was initially characterized in 2008 through molecular phylogenetic analysis of the 16S ribosomal RNA (rRNA) gene, the 65kilodalton heat shock protein (hsp65) gene, and the secA1 gene, substantiated by DNA-DNA hybridization techniques.[8].To date, a total of nine documented clinical cases of infections caused by Nocardia wallacei have been globally reported [8][9][10][11][12][13][14][15][16].It has become one of the emerging species of human pathogenic Nocardia and has brought therapeutic challenges, which have attracted researchers' attention due to its high levels of antibiotic resistance, especially to the aminoglycosides.There are fewer types of antibiotics available for the treatment of Nocardia wallacei compared to other Nocardia spp. of antibiotic therapy [17].Trimethoprim-sulfamethoxazole alone or in combination is still the first line and preferred empirical medication for the treatment of all types of nocardiosis, including Nocardia wallacei infections, due to the high sensitivity of the Nocardia genus to sulfonamide [2,18].We report the first patient with Nocardia wallacei infection who was allergic to sulfamethoxazole and improved after treatment with linezolid in Zhejiang.

Case presentation 2.1 Clinical features
A 61-year-old female farmer was admitted due to persistent back pain.Abnormal elevation of neutrophils (79.2%) and C-reactive protein (46.5 mg/L) were indicated by blood routine examination (Table 1).Computed tomography (CT) scan showed pulmonary inflammation with bronchiectasis (Figure 1).
Informed consent: Informed consent has been obtained from all individuals included in this study.

Etiological examination
Microscopic characteristics of the patient's alveolar lavage fluid were determined by Gram staining.A large number of Gram-positive filamentous bacteria were observed to be arranged radially, with varying shades of staining resembling beads under oil microscopy, indicating that it may be of the genus Nocardia (Figure 2a).Acid-fast staining was weakly positive under an oil microscope (Figure 2b).White colonies of varying sizes, dry or waxy colonies, with wrinkles and particles on the surface were observed after 3 days of aerobic incubation on a blood agar plate (containing blood components that provide ample nutrients and growth factors, suitable for the growth of most aerobic and facultative anaerobic bacteria) of alveolar lavage fluid (Figure 2c).The Nocardia wallacei colony was picked out with sterile toothpicks or inoculation rings, and the bacteria were evenly smeared on the target plate dedicated to Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to form a thin layer to avoid overlapping.Formic acid was added dropwise, and the plate was dried.Matrix liquid was then added dropwise.After the target plate was dried, it was detected on the machine.In the mass spectrometer, the sample was separated by effective ionization to form a clear fingerprint of bacteria specificity.MALDI-TOF MS was used for colony identification and it indicated Nocardia wallacei with a 99.9% confidence (Figure 2d).

Treatment and prognosis
Due to the patient's complaint about previous allergies to trimethoprim-sulfamethoxazole, we used an intravenous infusion of 0.6 g of linezolid every 12 h for anti-infection treatment of Nocardia wallacei.After 18 days, the patient's inflammatory indicators returned to normal and the condition improved significantly.The intravenous injection of linezolid was stopped and the patient was discharged.She was recommended to continue taking oral linezolid for treatment and regularly return to the respiratory clinic for follow-up.The patient's current prognosis is excellent according to our latest follow-up (6 months since the first treatment of linezolid).

Discussion
The clinical manifestations and imaging features of nocardiosis are nonspecific, so it may be misdiagnosed as other infections including lung abscess, tuberculosis, bacterial pneumonia, and pulmonary aspergillosis [19].Pulmonary infection is the most common manifestation of nocardiosis [20].The lung CT of our case showed inflammation, and the lung infection was likely acquired by inhaling Nocardia from dust or soil.The patient is a farmer who has been working in the field for a long time, so she may have the potential risk of infection by pathogens in the soil.Although Nocardia has been reported in medical literature, Nocardia wallacei infection in humans is rare and easily   [21].MALDI-TOF MS has become the reference method for routine identification of bacterial isolates in clinical microbiology laboratories worldwide [22].MALDI-TOF MS analysis identified the isolate as Nocardia wallacei, thereby consolidating the diagnosis of a Nocardia infection.A German pathologist discovered in 1932 that berberine had an inhibitory effect on Streptococcus, marking the earliest discovery of the antimicrobial effect of sulfonamide drugs [15].Sulfonamide drugs have been in clinical use for nearly 50 years, exhibiting advantages such as a broad spectrum of antibacterial activity, stability, low cost, high production yield, and convenient usage [23].The combination of trimethorprim-sulfamethoxazole remains one of the preferred antibiotic treatments for Nocardia infections [24,25].In this case, the patient exhibited an allergic reaction to sulfonamide drugs, rendering them unsuitable for treatment.Consequently, we opted for linezolid to improve the patient's condition.Linezolid is a synthetic antimicrobial agent, belonging to the oxazolidinone class of antibiotics.It finds clinical utility in treating infections caused by Gram-positive aerobic bacteria including Nocardia wallacei [14].Masahiro Toyokawa et al. found in their study of 146 strains of Nocardia receiving AST that linezolid was the most active drug among all species and did not exhibit in vitro resistance.In the absence of species identification, linezolid is a preferential empirical treatment option [16].This is the first case report of Nocardia wallacei infection in Zhejiang, China, and it confirmed that it can cause pulmonary infection in immunocompetent patients.

Conclusion
We report the first case of pulmonary infection caused by Nocardia wallacei in a patient with normal immune function in Zhejiang province.This Nocardia wallacei isolate was successfully identified by MALDI-TOF MS, which provided a new option for the identification of Nocardia.After treatment with linezolid, inflammation indicators of the patient returned to normal and improved.Including this case, only 10 cases of Nocardia wallacei infection have been reported so far, thus more cases are needed for the investigation of spatiotemporal distribution and drug sensitivity of Nocardia wallacei isolates as well as clinical manifestation, morbidity and mortality of the patients.
Ethical approval: The research related to human use has complied with all the relevant national regulations, and institutional policies and in accordance with the tenets of the Helsinki Declaration, and has been approved by the Ethics Committee of Haiyan People's Hospital.(Ethics Committee Approval of Biomedical Research Involving Humans, Approval No.: 202310).

Figure 1 :
Figure 1: CT image of the patient's lungs.

Figure 2 :
Figure 2: Etiological examination: (a) Gram-positive bacteria were observed by Gram staining under an oil mirror (×1,000 magnification); (b) acid-fast staining under oil mirror (×1,000 magnification); (c) wrinkled, dry, and white colonies of different sizes were observed by culture on the blood agar plate; (d) MALDI-TOF MS confirmed it was Nocardia wallacei.

Table 1 :
Clinical and laboratory indicators of the patient

Table 2 :
Reported cases of N. wallacei in immunocompetent and immunocompromised patients

Table 2 :
[8] total of nine cases of Nocardia wallacei infection were reported in the world, including four cases of immunocompetent patients and five cases of immunocompromised patients (one case of diabetes mellitus, two cases of HIV infection, one case of lymphoma, and one case of oral carcinoma; Table2).Nocardia is identified as a bacterium that is aerobic, gram-positive, partially acid-fast, resistant to lysozyme, and positive for catalase[8].It displays a unique cellular morphology with beaded, branching characteristics.Diagnosis and identification of Nocardia are crucial due to the requirement for specific antibiotics in treatment, with antibiotic susceptibility varying among strains.Initially, careful examination of pus, sputum, or other exudates and secretions for pigment granules is conducted.Smears or crush preparations of these samples undergo Gram staining and acid-fast staining.The presence of Gram-positive, branching filaments that are partially acid-fast may suggest Nocardia, albeit requiring differentiation from Mycobacterium tuberculosis.In this case, upon bronchoalveolar lavage (BAL) fluid smear microscopy, numerous Gram-positive, filamentous bacteria arranged radially were observed, appearing like beads under oil immersion microscopy with variations in staining intensity, indicative of possible Nocardia species.A weakly positive result on modified acid-fast staining further supported this preliminary identification.Culturing of the BAL fluid yielded colonies of varied sizes, with a wrinkled, dry, and white appearance.In recent years, the number of microbial species that MALDI-TOF MS can routinely identify has increased, and now, it can reliably identify closely related species, including Nocardia misdiagnosed