Disseminated infection with Nocardia otitidiscaviarum in a patient under steroid therapy

Key Clinical Message Corticosteroid recipients with lung infections should be suspected of having nocardiosis; however, nocardiosis can easily mimic malignancy, tuberculosis, or fungal infection. Though cultural identification is possible, it might be missed due to its slow growth pattern.. Therefore, if filamentous bacteria are seen during staining, plate incubation time should be extended.


| BACKGROUND
Nocardia otitidiscaviarum is a rare pathogen and is known to be less pathogenic than other Nocardia species.
The genus Nocardia is a ubiquitous group of environmental bacteria found in soil, decomposing vegetation, and other organic matter, as well as in fresh and salt water. 1 It usually manifests as an opportunistic infection in immunocompromised hosts. It is Gram-variable, acid-fast, and branching filamentous bacteria which grows aerobically. In humans, the infection occurs either by inhalation of dust or contamination of wound or by deep implantation. Hence, Nocardia can cause pulmonary, superficial cutaneous, and subcutaneous infection (mycetoma). 2 Nocardia infection can be caused by various species, including N asteroides, N brasiliensis, N otitidiscaviarum, N cyriacigeorgica, and N farcinica. 3 Out of all Nocardia species, N asteroids, N farcinica, and N brasiliensis are the primary pathogens causing nocardiosis, while other species are rare or reported less frequently. 4 N otitidiscaviarum rarely causes infection, and it was first recognized in samples taken from a Sumatran cavy or guinea pig with ear disease. 3 It is usually less pathogenic than other species of Nocardia. 5,6 Herein, we present a case of N otitidiscaviarum, a rare and less pathogenic organism causing severe pulmonary and lymphocutaneous (subcutaneous abscess) infection.

| CASE PRESENTATION
A 61-year-old man presented to Tribhuvan University Teaching Hospital (TUTH), Kathmandu, Nepal, with chief complaints of fever for seven days along with coughing and swelling of the right thigh. Fever was continuous, with maximum recorded temperature of 102 degree Fahrenheit. There was no history of night sweats, hemoptysis, weight loss or abdominal swelling. The patient did not report any history of trauma. There was no contact history with tuberculosis patient. For his illness, he was treated with intravenous ceftriaxone at a local hospital for 10 days; however, his symptoms did not improve and was referred to TUTH.
He was on steroid (1 mg/kg/d) therapy since 8 weeks for his recent diagnosis of nephrotic syndrome (focal segmental glomerulosclerosis) on renal biopsy. On clinical examination, a mass was palpated over his right thigh. His cardiovascular, neurological, and gastrointestinal examinations were unremarkable. On respiratory examination, patient was tachypneic, dull note was heard over right upper hemithorax, and breath sound was deceased on right upper hemithorax.
On laboratory examination, his total white blood cell (WBC) count was 16 000/µL with neutrophilic predominance (82%). Erythrocyte sedimentation rate (>56 mm/h) and C-reactive protein (112 mg/L) were elevated with normal procalcitonin (0.4 ng/mL) level. His serum creatinine (0.9 mg/dL) and liver enzymes were within normal range. Serum markers for HIV, hepatitis B and hepatitis C, and syphilis were negative. Chest X-ray showed opacities. Highresolution computed tomography (HRCT) of chest showed consolidation (mass-like lesion 3.5 × 3.5 cm) in the right upper lobe with right-sided pleural effusion and cystic lesion in the left upper lobe (Figure 1). MRI of the brain revealed no neurological findings. Ultrasonography (USG) of the abdomen was normal but USG of thigh showed pus collection in the right thigh.
Gram staining of sputum sample showed Gram-variable branching filamentous bacteria. Modified Ziehl-Neelsen (ZN) staining of the same sample showed acid-fast branching filamentous bacteria ( Figure 2). Gene Xpert MTB/RIF test of sputum sample was negative. On aerobic culture, chalky-white colonies were seen on blood agar. The subcutaneous abscess was aspirated, and the pus was sent to laboratory for further examination. Gram stain revealed plenty of pus cells, with Gram-variable, branching, and filamentous bacteria. On the background of filamentous bacteria on Gram stain and ZN stain, modified ZN stain was performed. It revealed numerous acid-fast branching filamentous organism with beaded appearance ( Figure 3). The pus was cultured both aerobically on blood and MacConkey agar and anaerobically in Robertson's cooked meat media and incubated at 37°C. There was no growth in the agar plates at 24 hours of incubation, and no turbidity was seen in Robertson's cooked meat media. But on further incubation, adherent chalky-white colonies were seen on blood agar. On prolonged incubation, the colonies became yellowish and had molar tooth appearance ( Figure 4). These colonies were positive with catalase test and urea hydrolysis test. However, no growth was seen on MacConkey agar and in Robertson's cooked meat media which was incubated anaerobically.
For species identification, the isolated colonies were transported to India where VITEK ® MS was applied which uses matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) technology. As a result, Nocardia otitidiscaviarum was identified with confidence value of 99.9%. The colonies were processed for antimicrobial susceptibility testing by microbroth dilution method as per CLSI guidelines and were sensitive to cotrimoxazole, imipenem, amikacin, and linezolid but resistance to ceftriaxone.
His respiratory distress increased, leading to high oxygen demand. Therefore, he was shifted to ICU and intubated. His weight was 58 kg, height 5.7″, and BMI 19.9. So meropenem (1 gm three times daily) and amikacin (750 mg once daily) were initiated in addition to cotrimoxazole. Arterial blood gas analysis revealed pH-7.491, Pao2-51.9 mm Hg, Pco2-30.5 mm Hg, and Hco3-23.5 mmol/L with Pao2/ Fio2-86.4 mm Hg. After 7 days, his fever resolved with a decrease in WBC count and improvement in respiratory symptoms. He was shifted to general ward where amikacin (750 mg once daily) with cotrimoxazole (800/160 two tab twice daily) was continued and linezolid (600 mg twice daily) was added to the regimen for 21 days as induction therapy.
Meropenem was switched to linezolid in view of species identified as N otitidiscaviarum which is less susceptible to beta-lactam antibiotics.
He was discharged with cotrimoxazole for 6 months as maintenance therapy. Chest X-ray was done at 1, 3, and 6 months ( Figure 5) which showed progressive resolution of the lesion. He was on regular follow-up without any drug reaction or complication. There was no relapse of symptoms at one-year follow-up.

| DISCUSSION
To the best of our knowledge, this is the first case of N otitidiscaviarum infection in Nepal. Few cases of Nocardia infection of brain, lung, eye, and soft tissue have been reported from Nepal but none of them had performed genetic analysis and identification up to species level. Snijders first isolated N otitidiscaviarum in 1924, 7,8 and Gordon et al first identified its biochemical reactions and distinguished it from other Nocardia species. 8 Nomenclature of N caviae was originally proposed, but now the organism is called N otitidiscaviarum. 8,9 A study performed at the National Reference Laboratory in Germany from 1979 to 1991 isolated Nocardia spp. form 131 patients; however, only 8 of them were infected with N otitidiscaviarum. 8 N otitidiscaviarum caused much less infections than other Nocardia, which may be due to its lower prevalence in soil and its reduced pathogenicity. 8 Despite its low prevalence and pathogenicity, sometimes like our case, it can lead to severe infection. Nocardia otitidiscaviarum has been described as an opportunistic pathogen in human. 10,11 However, it has been reported in both immunocompromised and immunocompetent individuals causing pulmonary, primary cutaneous, and lymphocutaneous infections. 11,12 Individuals with weak immune system, such as patients suffering from chronic obstructive pulmonary disease, diabetes mellitus, mixed connective tissue disorder, ulcerative colitis, cirrhosis, human immunodeficiency virus infection, malignancies, those receiving long-term corticosteroid therapy, and bone marrow or solid organ transplant, are at higher risk. 11,13 Nocardia infection has nonspecific pathogenic signs or symptoms, and its clinical picture may mimic a variety of other bacterial infections including actinomycosis, tuberculosis, fungal infections, and malignancies. 14 A definitive diagnosis can be made by isolation and identification of the organism. For the initial evaluation, Gram stain and modified acid-fast stain must be done. 15 Although Nocardia spp. grow on ordinary blood agar, but its growth is slow (ranging from 2 days to weeks). Hence, the plates should be incubated for up to 2 or 3 weeks for slowly growing Nocardia species. 4

F I G U R E 3 Modified Ziehl-Neelsen (ZN) stain of pus showing
acid-fast branching filamentous organism with beaded appearance

F I G U R E 4 Molar tooth appearance colonies of
Nocardia otitidiscaviarum on blood agar at prolonged incubation The drug choice for Nocardia infection is cotrimoxazole, but some of the species are resistance to cotrimoxazole, imipenem, amoxicillin-clavulanic acid, and other antibacterial agents (Table 1) [14][15][16][17][18][19][20][21][22] ; therefore, accurate identification at species level and antimicrobial susceptibility testing must be done. 19 N otitidiscaviarum infection requires treatment for long duration, and also, it is suggested that antibiotic therapy should be continued for 6 months in immunocompetent patients and up to a year in immunosuppressed individuals. 11 Most N otitidiscaviarum isolates are resistant to beta-lactams like imipenem, ampicillin, and amoxicillin-clavulanic acid but are susceptible to fluoroquinolones and amikacin. 5,11 Meanwhile, some other studies have shown that N otitidiscaviarum complex is sensitive to linezolid in vitro; however, data from in vivo studies are lacking. In addition, 4 weeks of linezolid treatment increases the risk of hematological toxicity, and also, clinical experience with linezolid treatment is limited. 11,20 In our case, we prescribed 3 weeks of linezolid in combination with cotrimoxazole and amikacin as induction therapy followed by maintenance with cotrimoxazole for six months.
Minocycline can be used as an alternative agent when sulfa drugs cannot be given. Imipenem and amikacinin higher doses have been used in severe or refractory cases. 20 Other alternative antimicrobial agents include amoxicillin-clavulanic acid, ceftriaxone, cefotaxime, meropenem, linezolid, moxifloxacin, levofloxacin, and tigecycline can be also used. To minimize the risk of relapse, the treatment is generally prolonged. 1 The US National Committee of Clinical Laboratory Standards has approved broth microdilution as antimicrobial susceptibility testing for both Actinomycetes and Nocardia. 11 This may guide the treatment of N otitidiscaviarum, but the optimal treatment protocol of N otitidiscaviarum is still unknown. A combination of sulfonamides and amikacin with carbapenem or third generation cephalosporin had been suggested for severe or disseminated infections. 11 Similarly, in this case, the combination therapy (amikacin, meropenem, and cotrimoxazole) was administered empirically and then modified to amikacin, cotrimoxazole, and linezolid after identification of the species.
Nocardia otitidiscaviarum is isolated rarely; however, it can cause localized or disseminated infection, even in an immunocompetent host. The majority of patients responds to cotrimoxazole and aminoglycoside. Identification and drug susceptibility testing for Nocardia species are critical for guiding clinical treatment. 8 F I G U R E 5 Chest X-ray at 6 mo showing resolution of the pulmonary lesion T A B L E 1 Antimicrobial susceptibility patterns for common Nocardia species [14][15][16][17][18][19][20][21][22]

| CONCLUSION
Diagnosis of Nocardia infection is often misleading and can be initially diagnosed as malignancy or other bacterial (eg, tuberculosis) or fungal infection due to its clinical resemblance. It is usually missed in routine culture because of its slow growth. Therefore, laboratory should keep the culture plate for prolonged incubation whenever filamentous bacteria is seen in Gram stain or modified ZN stain. Physicians should suspect Nocardia infection as differential diagnosis in patient receiving steroids with pulmonary lesions. Species identification and antibiotic susceptibility testing are required for guiding antibiotic therapy for Nocardia infection, and the patient should be treated with prolonged antibiotic therapy for complete cure and prevention of relapse.