Folia Microbiol DOI 10.1007/s12223-013-0298-7

Pulmonary nocardiosis associated with cerebral abscess successfully treated by co-trimoxazole: a case report Seyyed Saeed Eshraghi & Siamak Heidarzadeh & Abdolreza Soodbakhsh & Mohammadreza Pourmand & Amir Ghasemi & Mohsen GramiShoar & Ensieh Zibafar & Amir Aliramezani

Received: 15 April 2013 / Accepted: 16 December 2013 # Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2014

Abstract Nocardiosis is an acute or chronic infectious disease caused by the soil-borne filamentous bacteria belonging to the genus Nocardia. The organisms opportunistically infect both immunocompromised and immunocompetent individuals. The lungs are the primary site of infection and brain abscess is, by far, the most common complication following nocardial metastasis from pulmonary lesions. Although surgical intervention must always be considered in the treatment of nocardial brain abscess, it can obviously be cured by antibiotic therapy alone. This report describes a case infected by Nocardia cyriacigeorgica. Identification of the infectious agent was achieved by conventional and semi-nested PCR techniques. A 55-year-old woman with fever was referred to the infect disclinic of Imam Khomeini hospital in Tehran and was hospitalized after clinical assessment. She was a kidney transplant recipient for 4 years and was taking immunosuppressive treatment including azathioprine and methylprednisolone. Followup of the patient by CT scan revealed pulmonary infection and cerebral lesions. Specimens of the brain lesions contained filamentous bacteria. The patient received a combination of cotrimoxazole and ceftriaxone and brain abscesses as well as lung S. S. Eshraghi (*) : S. Heidarzadeh : M. Pourmand : A. Ghasemi : A. Aliramezani Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran e-mail: [email protected] A. Soodbakhsh Department of Infectious Diseases, Imam Khomeini Hospital Complex, Tehran University of Medical Sciences, Tehran, Iran M. Pourmand Biotechnology Research Center, Tehran University of Medical Sciences, Tehran, Iran M. GramiShoar : E. Zibafar Department of Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

inflammation disappeared gradually during the course of antibiotic therapy within 3 months. The patient was discharged from the hospital after 2 months of therapy. Keywords Nocardia cyriacigeorgica . Brainabscess . Kidney transplant . 16S rRNA

Introduction Nocardia species are ubiquitous saprophytes which can be found in soil, water, decaying plant materials, dust, and air. They occasionally cause disease in humans, especially in immunosuppressed patients (Eshraghi et al. 2009). Pulmonary involvement is observed in 73–77 % of infected cases because the lungs are the primary portal of entry. Studies have shown that nocardiosis is significantly associated with morbidity and mortality of patients with kidney transplant who receive immunosuppressive therapy (Batista et al. 2011). In rare cases, lung or skin infections may spread to other organs such as brain, kidney, and liver, leading to serious infections in these sites (Batista et al. 2011). The cerebral tissue is more susceptible to Nocardia species, and the brain is most likely to be the target organ in hematogenous dissemination consequent to pulmonary nocardiosis (Rakotoarivelo et al. 2011). Although nocardial brain abscesses are rare, they are the most common non-pulmonary lesions in disseminated nocardiosis and have been reported in 10–15 % of patients with this infection. Usually, the mortality rate of brain abscesses is estimated to be about 10 %, but cerebral nocardiosis shows a death rate of 30 % because about half of the affected patients are immunosuppressed (Duran et al. 2001; Malincarne et al. 2002). Early diagnosis and management significantly reduces the morbidity and mortality rates of nocardiosis (Mijares and Mendoza 2001). Since the identification of nocardial cerebral abscesses is difficult to deal with, detection of the specific etiologic agent is

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essential for rapid and efficient treatment. Due to lack of discriminating clinical signs, most of the cases could not be recognized in due time (Couble et al. 2005) Yassin et al. described Nocardia cyriacigeorgica for the first time in 2001 (Yassin et al. 2001) which has been classified in Nocardia asteroides drug pattern type VI. The study of Wallace et al. (1988) has shown that N. asteroides displays six drug resistant patterns (Brown-Elliott et al. 2006) and N. cyriacigeorgica has been proposed to be the most commonly isolated drug pattern type of N. asteroides in southern USA. Also, N. cyriacigeorgica infections have been reported in Canada, North America, Europe, and Asia (Barnaud et al. 2005; Cloud et al. 2004; Elsayed et al. 2006; Maraki et al. 2006; Brown-Elliott et al. 2006). In the present study, we aim to report a case of a primary cerebral abscess due to N. cyriacigeorgica in an immunocompromised woman who had received kidney transplantation.

Materials and methods Case study A 55-year-old woman with history of fever admitted to the hospital. She had received a kidney transplant 4 years before and was receiving immunosuppressive drug, azathioprine, and methylprednisolone. Since she had symptoms of cytomegalovirus (CMV) infection including diarrhea and vomiting, a colonoscopy was performed to help diagnosis. A negative colonoscopy result ruled out cytomegalovirus infection. The patient was released from the hospital with certain dosages of ciprofloxacin and methylprednisolone. Patient was febrile for 24 h; after discharge, she returned to the hospital with high fever and asthma. Radiographic testing was used to determine the presence of pulmonary embolism or any lung infections. Chest x-ray indicated several small caverns of the lung. Echocardiogram was carried out to check the probability of endocarditis. The patient did not show any signs of cardiac dysfunction. Blood and urine cultures were negative. To evaluate the existence of nocardial or mycobacterial pulmonary infections, bronchoscopy was carried out and bronchoalveolar lavage (BAL) samples were collected and transferred to the microbiology laboratory at the Tehran University of Medical

Sciences for further investigations. Although culture results from BAL was not positive, further investigation, including molecular methods, suggested that the isolated bacterium might be from the Nocardia species. Upon detection of infections, the patient was under co-trimoxazole, imipenem, and vancomycin with specific doses of treatment. Since echo results and blood/urine cultures were negative, vancomycin treatment was discontinued. MRI and CT scan observation indicated multiple brain and lung abscesses (Figs. 1, 2, and 3). Surgical consultation plus the presence of filamentous bacteria in clinical samples indicated the possibility of nocardial infection. The brain lesion samples were also sent to the diagnostic microbiology laboratory for further investigation. Considering the critical situation of the patient (such as hypoxia, hypotension, tachypnea, and impaired consciousness), she was hospitalized in the ICU and ceftriaxone was added to her treatment regime. After 10 days, her overall health status started to improve daily. Upon follow-up of the patient, CT scan showed reduction of the brain lesions. Brain CT scan demonstrated that a huge number of large cerebral lesions had become smaller and preexisting smaller lesions of the brain were vanished. The patient gained her consciousness gradually after 1-month treatment with co-trimoxazole. Lung CT scan indicated complete clearance of the lungs after 1 month of treatment. The patient was discharged from the hospital 3 months after taking cotrimoxazole and continued receiving co-trimoxazole treatment. After 90 days, brain lesions disappeared. Isolated bacteria and growth conditions Gram and modified kynion acid fast staining of the smears revealed Gram-positive, partially acid fast branching organisms. The brain biopsy was cultured onto Löwenstein-Jensen, blood agar, and Sabouraud dextrose agar, and incubated at 37 and 45 °C. Organisms grew after 4 days of incubation in all six media. Primary observation of colonial morphology determined the typical colonies of Nocardia species. Further identification of the bacteria were carried out by using biochemical tests including hydrolysis of casein, tyrosine, xanthine, hypoxanthine, urease, growth at 45 °C, and utilization of

Fig. 1 MRI of multiple brain abscess in a 55-year-old woman who had received kidney transplantation

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15 min at 95 °C. DNA was extracted with MTB respiratory specimen preparation kit (Roche, Germany), according to the manufacturer’s instructions (Couble et al. 2005). PCR

Fig. 2 CT scan of lung abscesses in the nocardiosis case

acetamide. All the tests were negative, except growth at 45 °C and utilization of acetamide. Antibacterial susceptibility tests were performed using disk diffusion method on MuellerHinton agar (Wallace et al. 1988). The organism was susceptible to, imipenem, amikacin, cefotaxime, trimethoprimsulfamethoxazole, and linezolid but was resistant to ciprofloxacin, ampicillin, penicillin G, vancomycin, ciprofloxacin, erythromycin, amoxicillin-clavulanic acid, clarithromycin, and kanamycin after 2-days incubation at 37 °C.

Molecular methods DNA extraction Pure bacteria were cultured in blood agar at 37 °C for 72 h. Colonies were picked off with a loop, and one loopful of bacterial cells was suspended in 250 μL of PBS and 50-μL proteinase K (Sigma Aldrich, Saint Quentin Fallavier, France), and then mixed up by vortex for 1 min. The mixture was then incubated for 24 h at 37 °C. Proteinase K was inactivated for

The 598-bp fragment of 16S rRNA was amplified using primers NG1(5′- ACCGACCACAAGGGGG-3′) and NG2 (5′-GGTTGTAAACCTCTTTCGA-3′). The sequences of the primers were based on the study of Laurent et al. (1999). The PCR mixture contained 2.5-μL PCR buffer, 1-μL MgCl2, 1 μL of each dNTP, 1 μL of each primer, 1 U of Taq DNA polymerase, and 5 μL of the extracted DNA in a 25-μL final volume. PCR amplification was performed in a DNA thermal cycler (MJ Research Inc., MA, USA) and included initial denaturation at 95 °C for 5 min, and then subjected to 30 cycles of amplification (denaturation at 95 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 2 min) followed by a final extension step at 72 °C for 10 min. PCR products were visualized by 1 % agarose gel electrophoresis and ethidium bromide staining. Cloning and Sequencing Five microliters of the extracted DNA was used in the 50-ml PCR mixture. The PCR mixture contained 5 μmol/L PCR buffer, 5 μmol/L MgCl2, 1 μmol/L each dNTP, 2 μmol/L each primer, and 1 U of high fidelity Taq polymerase (Fermentas, Latvia). The PCR conditions entailed 5 min of denaturation at 95 °C, followed by 30 of amplification cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 2 min, and ended with a final extension at 72 °C for 30 min. A 992-bp fragment of the 16S rRNA gene was amplified with primers described by Conville et al. (5′-CGAACGCTGG CGGCGTGCTTAAC-3′) and (5′-ACCTGTACACCGACCA CAAGGGGG-3′) (Conville et al. 2000). The purified fragment was cloned into plasmid pTZ57R/T, and then used to transform Escherichia coli strain TOP10F′, using the reagents and protocols supplied by the instructions of Fermentas InsTAclone™ PCR Cloning Kit (Fermentas, Latvia) (Brown et al. 2004). Plasmid DNA containing the PCR fragment was purified by using the protocol and reagents supplied with the Plasmid Mini Extraction Kit protocol (Bioneer, Korea) (Brown et al. 2004). The extracted plasmid was used for sequencing. Sequencing result was analyzed by DNAMAN software using Nocardia species, sequences from GenBank.

Results and discussion

Fig. 3 CT scan of brain abscesses in the nocardiosis case

The results of MRI observation demonstrated multiple brain abscesses in patient (Fig. 1). CT scan of lung also revealed lung abscess (Fig. 2). Brain CT scan revealed several ring enhancing lesions of the brain (Fig. 3).

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In addition to conventional methods which are based on phenotypic, biochemical, and enzymatic profiling, molecular methods were used to identify Nocardia in this study. The nucleotide sequencing results demonstrated a 100 % homology to that of the 16S rRNA of N. cyriacigeorgica (Fig. 4). Despite the fact that culture is still the “gold standard” method when bacterial central nervous system infections are involved, several technical problems are associated with this method. The downsides of bacterial culture include antibiotic therapy, small sample volumes, suboptimal duration of incubation, and suboptimal growth medium (McNeil and Brown 1994). Compared to genotypic method, diagnosis of nocardiosis based on conventional phenotypic characterization such as chemotaxonomic, biochemical tests, and morphology is very time-consuming, laborious, and expensive. In addition, mis-identification of Nocardia species may occur considering the presence of closely related bacteria including Dietzia, Gordonia, Mycobacterium, Rhodococcus, Tsukamurella, and Streptomyces (Laurent et al. 1999). Some Nocardia species are non-cultivatable and others grow slowly. Therefore, diagnosis of Nocardia infections by culture can take 1–2 weeks, or the culture can be overgrown by a contaminating organism before Nocardia grows (Tatti et al. 2006). Studies indicated that empirical antibiotic therapy administered before sampling can also prevent or postpone the growth of bacteria, especially in cerebral abscesses (Couble et al. 2005). Due to the time-consuming nature of phenotypic methods, nocardiosis is infrequently diagnosed after the dissemination of the strain to other organs or after the patient has died. Therefore, it is difficult to initiate therapy at the early stages of the disease (Couble et al. 2005; Reis et al. 1995).

Because of the increasing resistance of Nocardia species to antibiotics and due to the lengthy time for cultivation and the potential overgrowth by other organisms in culture, it is necessary to use fast and sensitive assays to diagnose infections caused by this pathogen (Tatti et al. 2006). The use of molecular approaches has turned out to be sensitive, specific, less time-consuming, and less labor-intensive than traditional methods (Brown et al. 2004). The molecular techniques eliminate the above mentioned problems by the specific amplification of nocardial DNA, even if several other contaminating or colonizing bacteria are present in the specimens. The direct diagnosis of nocardiosis by PCR of cerebrospinal fluid could substitute the use of cerebral biopsy (Laurent et al. 1999). The PCR assay can differentiate Nocardia species from each other and closely related bacteria quickly and meticulously. PCR assay may simplify the identification of new emerging bacterial pathogens to enable an early diagnosis and enhanced outcome in therapy. It also provides a beneficial means device for screening multiple specimens quickly (Brown et al. 2004). The accessibility of gene sequencing has revolutionized the taxonomy of the genus Nocardia and has become a valuable means for the identification of clinical isolates of this genus. Gene sequencing provides fast and generally reliable identification of Nocardia isolates. It has become obvious that the number of distinct taxa and the number of species capable of causing human disease were underestimated when identification relied solely on phenotypic characteristics (Roth et al. 2003). 16S rRNA, hsp65, and secA1 genes have been used for identification purposes, but nowadays, no sequence information for hsp65 and secA1 genes for Nocardia species are available in public databases. The 16S rRNA gene is most often used for species identification (Brown-Elliott et al. 2006). Since 16S rRNA gene sequencing is often used for species identification, isolates with unique 16S rRNA gene sequences have been designated distinct species, resulting in a considerable increase in the number of recognized species of Nocardia (Brown-Elliott et al. 2006).

Strain identification

Fig. 4 Polymerase chain reaction (PCR) analyses of 16S ribosomal DNA (rDNA) in brain abscess specimen. Agarose gel electrophoresis of PCR product by using up-stream and down-stream primers. Amplification of the 992-bp fragment of the 16S rRNA gene

In the present study, we used PCR technique to amplify sequence and a segment of the 16S rRNA gene. Our results indicate that the specific Nocardia type in our case was N. cyriacigeorgica. Antibiotic therapy should be initiated once the antibiotic susceptibility pattern of the bacteria is identified. In this case, the strain was sensitive to co-trimoxazole (Berkey and Moore 1998). Three-month treatment with this antibiotic resulted in complete disappearance of brain lesions. Cotrimoxazole is still the selected drug in the treatment of nocardial infection in renal transplant recipients (Mijares and

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Mendoza 2001). There is, however, reports of antimicrobial resistance and insufficient compliance brought about by gastrointestinal, hematologic, and allergic adverse effects (Wilson et al. 1989; Threlkeld and Hooper 1997; Leitersdorf et al. 1997). More importantly, impairment of renal function brought about by the interaction of cyclosporine and sulfonamides may occur in as much as 75 % of renal transplant recipients. So, it is very important to find alternate drugs to ensure adequate antimicrobial coverage in lieu of long-term treatment. Cephalosporins, imipenem, and meropenem are used as intravenous alternatives, while minocycline and coamoxyclavappear to be adequate oral substitutes of sulfonamides (Arduino and Johnson 1993). This is the first report of N. cyriacigeorgica in Iran associated with brain abscess in a kidney transplant recipient, and successfully treated with cotrimoxazole. Acknowledgments This study was supported by School of Public Health and Biotechnology Research Center, Project number 13434, Tehran University of Medical Sciences. The authors declare that they have no conflict of interests.

References Arduino RC, Johnson PC (1993) Nocardiosis in renal transplant recipients undergoing immunosuppression with cyclosporine. Clin Infect dis 16(4):505–512 Barnaud G, Deschamps C, Manceron V, Mortier E, Laurent F, Bert F, Boiron P, Vinceneux P, Branger C (2005) Brain abscess caused by Nocardia cyriacigeorgica in a patient with human immunodeficiency virus infection. J Clin microbiol 43(9):4895–4897 Batista MV, Pierrotti LC, Abdala E, Clemente WT, Girao ES, Rosa DR, Ianhez LE, Bonazzi PR, Lima AS, Fernandes PF, Padua-Neto MV, Bacchella T, Oliveira AP, Viana CF, Ferreira MS, Shikanai-Yasuda MA (2011) Endemic and opportunistic infections in Brazilian solid organ transplant recipients. Trop Med Int Health 16(9):1134–1142 Berkey P, Moore DKR (1998) In vitro susceptibilities of Nocardia species to newer antimicrobial agents. Antimicrob Agents Ch 32:1078–1079 Brown JM, Pham KN, McNeil MM, Lasker BA (2004) Rapid identification of Nocardia farcinica clinical isolates by a PCR assay targeting a 314-base-pair species-specific DNA fragment. J Clin Microbiol 42(8):3655–3660 Brown-Elliott BA, Brown JM, Conville PS, Wallace RJ Jr (2006) Clinical and laboratory features of the Nocardia spp. based on current molecular taxonomy. Clin Microbiol Rev 19(2):259–282 Cloud JL, Conville PS, Croft A, Harmsen D, Witebsky FG, Carroll KC (2004) Evaluation of partial 16S ribosomal. DNA sequencing for identification of nocardia species by using the MicroSeq 500 system with an expanded database J Clin Microbiol 42(2):578–584

Conville PS, Fischer SH, Cartwright CP, Witebsky FG (2000) Identification of nocardia species by restriction endonuclease analysis of an amplified portion of the 16S rRNA gene. J Clin Microbiol 38(1):158–164 Couble A, Rodriguez-Nava V, de Montclos MP, Boiron P, Laurent F (2005) Direct detection of Nocardia spp. in clinical samples by a rapid molecular method. J Clin microbiol 43(4):1921–1924 Duran E, Lopez L, Martinez A, Comuñas F, Boiron P, Rubio MC (2001) Primary brain abscess with Nocardia otitidiscaviarum in an intravenous drug abuser. J Med Microbiol 50:101–103 Elsayed S, Kealey A, Coffin CS, Read R, Megran D, Zhang K (2006) Nocardia cyriacigeorgica septicemia. J Clin Microbiol 44:280–282 Eshraghi SS, Talebi M, Namaki S, Mirshafiey A (2009) Nocardia. J Chinese Clin Med 4:48–66 Laurent FJ, Provost F, Boiron P (1999) Rapid identification of clinically relevant Nocardia species to genus level by 16S rRNA gene PCR. J Clin microbiol 37(1):99 Leitersdorf I, Silver J, Naparstek E, Raveh D (1997) Tetracycline derivatives, alternative treatment for nocardiosis in transplanted patients. Clin nephrol 48(1):48–51 Malincarne L, Marroni M, Farina C, Camanni G, Valente M, Belfiori B, Fiorucci S, Floridi P, Cardaccia A, Stagni G (2002) Primary brain abscess with Nocardia farcinica in an immunocompetent patient. Clin neurol and neurosurg 104(2):132–135 Maraki S, Panagiotaki E, Scoulica EM, Miari N, Hainis K, Dotis G, Katsoula I, Tselentis Y (2006) Nocardia cyriacigeorgica pleural empyema in an immunocompromised patient. Diag Microbiol Infect Dis 56:333–335 McNeil MM, Brown JM (1994) The medically important aerobic actinomycetes: epidemiology and microbiology. Clin Microbiol Rev 7(3): 357–417 Mijares MCC, Mendoza MT (2001) Pulmonary nocardiosis in renal transplant recipients. Phil J Microbiol Infect Dis:144-152 Rakotoarivelo RA, Raveloson HF, Razafimahefa SH, Farbos S, Gemain MC, Bonnal F (2011) Brain abscess with Nocardia asteroides revealing lung adenocarcinoma. Rev Pneumol Clin 67(5):329–330 Reis MA, Costa RS, Ferraz AS (1995) Causes of death in renal transplant recipients: a study of 102 autopsies from 1968 to 1991. J Royal Soc Med 88(1):24–27 Roth A, Andrees S, Kroppenstedt RM, Harmsen D, Mauch H (2003) Phylogeny of the genus Nocardia based on reassessed 16S rRNA gene sequences reveals underspeciation and division of strains classified as Nocardia asteroides into three established species and two unnamed taxons. J Clin Microbiol 41(2):851–856 Tatti KM, Shieh WJ, Phillips S, Augenbraun M, Rao C, Zaki SR (2006) Molecular diagnosis of Nocardia farcinica from a cerebral abscess. Hum pathol 37(8):1117–1121 Threlkeld SC, Hooper DC (1997) Update on management of patients with Nocardia infection. Curr Clin top infec dis 17:1–23 Wallace RJ Jr, Steele LC, Sumter G, Smith JM (1988) Antimicrobial susceptibility patterns of Nocardia asteroides. Antimicrob Agents Ch 32(12):1776–1779 Wilson JP, TURNER HR, Kirchner KA, Chapman SW (1989) Nocardial infections in renal transplant recipients. Medicine 68(1):38–57 Yassin AF, Rainey FA, Steiner U (2001) Nocardia cyriacigeorgica sp. nov. Int J syst evol microbiol 51(4):1419–1423