Nocardia Infection in Lung Transplantation

Organ transplant recipients (OTR) in general and, in particular, lung transplant recipients (LTx) – due to their underlying disease, extensive surgery, and the continual and profound immunosuppression to which they are subjected – become perfect targets for the development of infections. These are more frequent and severe than in the non-transplanted population, and may be caused by either common or less frequent opportunistic pathogens such as the Nocardia species.


Introduction
Organ transplant recipients (OTR) in general and, in particular, lung transplant recipients (LTx) -due to their underlying disease, extensive surgery, and the continual and profound immunosuppression to which they are subjected -become perfect targets for the development of infections.These are more frequent and severe than in the non-transplanted population, and may be caused by either common or less frequent opportunistic pathogens such as the Nocardia species.
Nocardia are ubiquitous bacteria found mainly in soil, organic matter and water (Lerner, 1996).Based on the available literature and on our own experience, we know that they can cause infections in both immunocompetent (40%) and immunocompromised patients (B.L. Beaman & L. Beaman, 1994), with a low frequency in transplantations in general , which is somewhat higher in LTx.Onset of disease is generally insidious and it is difficult to diagnose and treat.It produces mainly respiratory symptoms, but complications can occur, spreading to other organs and causing high mortality.In this chapter, we intend to review various epidemiological, clinical, diagnostic, therapeutic and prophylactic aspects of Nocardia infections in adult LTx.

General aspects
The genus Nocardia includes aerobic, gram-positive, weakly acid-fast bacteria of the order Actinomycetales.Other microorganisms that belong to this order are Corynebacterium, Rhodococcus, Gordonia, Tsukamurella, Actinomadura and Mycobacterium, particularly rapidly growing nontuberculous mycobacteria (Sorrell et al., 2005), which can also cause infections in humans.More than 50 species of Nocardia have been defined either phenotypically or by molecular methods (Brown-Elliott et al., 2006).Originally, only 10-12 species were known to cause disease in humans, the most frequently described being N. asteroides, N. nova, N. farcinica, N. transvalesis, N. brasiliensis, N. pseudobrasiliensis, N. otitidiscaviarum and N. brevicatena complex, with variations according to different environments and authors.Nowadays, taxonomy of this genus has undergone considerable changes due to modern molecular techniques, such as identification by 16S ribosomal RNA gene sequencing.New species have been described, such as N. cryacigeorgica (Schlaberg et al., 2008), N. veterana (Pottumarthy et al., 2003), N. abscessus (Yassin et al., 2000), N. paucivorans (Eisenblatter et al., www.intechopen.comLung Diseases -Selected State of the Art Reviews 394 2002) and N. kruczakiae (Conville et al., 2004), which should be taken into account in case of isolation in LTx samples.
Immune response to Nocardia is T-cell mediated (Deem, 1983).Therefore, the reduced cellular immune response of solid OTR is one of the major factors predisposing a patient to infection; in fact, 60% of cases occurs in immunocompromised patients.

Frequency
Estimated frequency of Nocardia infection among solid OTR globally ranges from 0.1 to 3.5 (Peleg et al., 2007a).In the past, these cases were more frequent in kidney, heart and liver transplantations, and less in LTx (Husain et al., 2002), but recently infections in the latter have been described as the most frequent (3.5%, Peleg et al., 2007a;1.8%, Santos et al., 2011;1.9%, Ponyagariyagorn et al., 2008).This increased risk of Nocardia infection in LTx may be due to several factors: a) the graft may have anatomical deficiencies, including lung denervation, reduced cough reflex and poor mucociliary clearance (Kramer et al., 1993;Husain et al., 2002); b) the organ has been continuously exposed to the atmosphere, with constant stimulation of the lung by environmental antigens and an increased risk of rejection; and, therefore, c) a need for more intense immunosuppression than in other solid OTR.Moreover, high average levels of calcineurin inhibitors 30 days prior to infection have been independently associated with subsequent infection by Nocardia (Hewagama et al., 2011).
The most frequently reported species of Nocardia in LTx, with variations in percentage in different studies, are N. nova, N. farcinica, N. asteroides and N. brasiliensis.

Risk factors
There are two multivariate studies (Peleg et al., 2007a;Martinez-Tomás et al., 2007) which investigate the influence of several variables as risk factors for Nocardia infection in solid OTR.Among them, three have been described as independent: a) the doses of immunosuppressive therapy, as already mentioned; b) chronic use of corticosteroids; and c) cytomegalovirus disease in the previous 6 months (Paya, 1999).
The relationship between opportunistic infections, including nocardiosis, and alemtuzumab, a monoclonal antibody that targets the antigen D52 and is used to prevent graft rejection (Basu et al., 2005;Peleg et al., 2007 b) by causing profound lymphopaenia, has been also reported.Moreover, the use of rituximab (Kundranda et al., 2007), another immunomodulator that targets the CD20 protein found on B cells and used in solid OTR to prevent or treat antibody-mediated rejection, has also been described.
Finally, hypogammaglobulinaemia, combined with immunosuppression, may favour the development of Nocardia infections (Corales et al., 2000), as has been observed in heart transplantation.Renal failure, prolonged respiratory support and early graft rejection may also be risk factors but have not yet been demonstrated in multivariate analysis.

Transmission
The main route for Nocardia infection is inhalation of aerosolised microorganisms.From the respiratory tract, bacteria can then move to other organs, the most commonly and seriously affected being the central nervous system (CNS).Involvement of skin and subcutaneous tissue, bones and joints, retina and, less frequently, other organs or structures (heart, kidney, peritoneum, endocardium, testicles, etc.) is also possible.Primary cutaneous infection can occur by direct inoculation in both immunocompetent and immunocompromised patients (Brown-Elliot et al., 2006).There is no evidence for human-to-human transmission and disease presents mainly as isolated cases, although an outbreak related to contamination by dust pollution (Sahathevan et al., 1991) in a liver transplantation unit and some spread by contamination of hands in a cardiovascular surgery unit (Wenger et al., 1998) have been documented based on molecular data.

Symptoms and radiology
Time of onset of infection after transplantation is variable, but tends to be late.It rarely occurs in the first month and may range from 1 to 28 years (Santos et al., 2011).The greatest risk is within the first year (Peleg et al., 2007a, Clark, 2009), but later cases have also been described (Peraira et al., 2003, Oszoyoglu et al., 2007).Pulmonary presentation is the most common, with subacute and insidious pneumonia.Less frequent is the cutaneous form, following a minor injury or by direct inoculation (Ambrosioni et al., 2010).Symptoms are usually non-specific and include fever, fatigue, dyspnoea, cough and pleuritic pain (Patel & Payá, 1997;Minero et al., 2009).Common radiographic abnormalities include irregular nodular lesions which may cavitate, diffuse interstitial infiltrates and lung consolidation with parapneumonic pleural effusion (Balikian et al., 1978;Morales et al., 2011) (Figure 1).Both lungs are usually affected, without significant anatomical or zonal distribution (Oszoyoglu et al., 2007).In the case of single-lung transplantation, Nocardia can infect both the native and the transplanted organ (Husain et al., 2002).Haematogenous spread has been reported in up to 50% of cases (Clark, 2009), so it is important to exclude Nocardia clinically and/or radiologically in other organs, especially in the CNS (Singh & Husain, 2000).Moreover, and especially when the patient has a central venous catheter, blood should be cultured to rule out Nocardia.Cerebral involvement may be asymptomatic, therefore requiring neuroimaging with magnetic resonance or high resolution computed tomography (Ambrosini et al., 2010), or symptomatic, with headache, vomiting, altered level of consciousness, focal signs and seizures.Multiple radiographic cerebral lesions are observed in 40% (Singh & Husain, 2000).Meningitis is uncommon.Other forms of presentation of dissemination are cutaneous, ocular, intestinal, testicular and bone and joint disorders.Primary as well as disseminated cutaneous forms may present as subcutaneous nodules, cellulitis, abscess, mycetoma and sporotrichoid skin changes (Merigou et al., 1998).In this location, N. brasiliensis is the most common.The presence of the cutaneous form in organ transplant recipients should be followed by the exclusion of other forms of presentation of nocardiosis.
We must take into account the possible and frequent co-infections that occur with common and opportunistic bacteria, viruses, especially CMV, and fungi, mainly Aspergillus (Cabada et al., 2010) that make the patient's clinical and therapeutic management difficult (Santos et al., 2011).Differential diagnosis of pulmonary infection and brain nodule must include Nocardia, Aspergillus spp., Cryptococcus neoformans, Mycobacteria, Rhodococcus equi, posttransplant lymphoproliferative disease and primary lung cancer with metastasis.

Microbiological diagnosis
Definitive diagnosis of nocardiosis requires microscopic observation, isolation or nucleic acid detection of Nocardia in one or more samples from a suspected site.Specimen collection can be spontaneous, such as in the case of sputum (which is useful in up to 53% of cases), superficial such as in the case of a skin smear, or may require deeper samples such as bronchoaspirate, bronchoalveolar lavage or tissue biopsies.Smear staining with Gram stain, Ziehl-Neelsen (ZN) and modified ZN is the most useful and fastest diagnostic method.It can provide a diagnosis within the first few hours, showing abundant gram-positive or partially acid-fast branched bacilli (Figure 2), which are very characteristic of Nocardia, with moderate or abundant leukocytes resulting from the inflammatory response.
Differential diagnosis with Rhodococcus, Gordonia and rapidly growing mycobacteria must be considered, as discussed above.This information provides a reliable presumptive diagnosis and may guide empiric antibiotic therapy.Although Nocardia may grow in non-selective culture media, samples that can be contaminated with normal flora, such as those from the respiratory tract, should also be cultured in selective media such as Thayer-Martin agar with antibiotics (Shawar et al., 1990).Typical colonies appear chalky white (Figure 3) with aerial hyphae.Its growth is aerobic and may take from 2 to 5 days, sometimes even a week.
Species identification may by phenotypic, at least for the most common Nocardia, but genotypic methods such as polymerase chain reaction (PCR), restriction endonuclease analysis and sequencing of a portion of the 16S rRNA gene provide a safer and more accurate diagnosis.These techniques, however, are carried out by a limited number of laboratories (Brown-Elliot et al., 2006).
Identification of the species can be useful to guide treatment, since some have intrinsic resistance to several antibiotics, or to predict prognosis, since some species, such as N. farcinica, are more virulent.In cases in which disseminated nocardiosis is suspected, Nocardia can also be isolated from a blood culture, although this is rare.

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Antimicrobial susceptibility testing of the Nocardia isolate is strongly recommended by the Clinical Laboratory Standard Institute (CLSI), which published the first approved methods in 2003 (Wayne, 2003).Primary susceptibility testing must include amikacin, amoxicillin/clavulanic acid, ceftriaxone, ciprofloxacin, clarithromycin, imipenem, linezolid, minocycline, trimethoprim-sulfamethoxazole (TMP/SMX) and tobramycin.Secondary recommendations include cefepime, cefotaxime, doxycycline, gentamicin and moxifloxacin.Some species of Nocardia have more predictable susceptibility patterns or, in other words, there are patterns of known resistance, such as N. otitidiscaviarum to imipenem, or the multiresistance of N. farcinica, so it is sometimes necessary to perform in vitro synergy studies.The susceptibility patterns of the most common Nocardia can be seen in table 1. Table 1.Expected and reported antimicrobial susceptibility of selected Nocardia species (% of isolates susceptible in various series).Composite data from references (Clark, 2009;Hewagama et al., 2011;Brown-Elliott et al., 2001).TMP/SMX, trimethoprimsulfamethoxazole; AMC, amoxicillin/clavulanic acid; CEF, ceftriaxone; IMP, imipenem; AMK, amikacin; LZD, linezolid; MIN, minocycline; CIP, ciprofloxacin; MXF, moxifloxacin; S, sensitive; R, resistant; V, variable.

General aspects
Treatment of Nocardia infections in LTx is based primarily on antibiotics.In some cases, such as brain or cutaneous abscesses that do not respond to initial antibacterial therapy, surgical drainage is also required and, whenever possible, especially in more severe cases, a reduction of immunosuppressive therapy should be applied.The best treatment has not yet been determined.In the past, the antimicrobial of choice was TMP/SMX, but since the development of new antibiotics, in vitro synergy studies, the insidious nature of the disease and its high mortality, the recommendation is a combination of two or three drugs (Clark, 2009).
There are few studies correlating susceptibility data with clinical outcome (Sorrell et al., 2005), so the best combination is uncertain.Nevertheless, it is often chosen on the basis of: a) the species of Nocardia isolated; b) its sensitivity to antibiotics and possible interactions with the complex medication of transplanted patients; c) the location, extent and severity of the infection; and d) the experts' opinion and documented experience in the literature.

Empiric therapy
According to clinical experience, TMP/SMX remains the antimicrobial agent of choice for many Nocardia infections (Hewagama et al., 2011), supported by the in vitro synergy of its two components, the fact that it reaches high levels in lung, brain, skin and (Smego et al., 1983) and that it can be used as intravenous or oral maintenance therapy (Table 2).The main side effects include rash, nausea, vomiting, erythema multiforme, bone marrow suppression, hyperkalaemia and crystalluria, which may limit its use.Some species -N.farcinica, N. otitidiscaviarum and N. nova -may be resistant to sulphonamides, so it is important to determine the species and its in vitro susceptibility.These resistances and possible allergies to sulphonamides promoted the search for alternative antibiotics.Amikacin is universally active against most Nocardia species, except for N. transvaliensis and N. brasiliensis.The main limitations for its use are optical and renal toxicity.The combination of imipenem and amikacin is accepted as initial therapy in patients with severe nocardiosis (Minero et al., 2009) while the antibiogram is pending.This antibiotic combination is additive and synergic in vitro (Kanemitsu et al., 2003), maintains synergy when associated with sulphonamides (Gombert et al., 1986) and is effective in humans, so this triple combination has also been recommended as first-line treatment in patients with severe disease including dissemination to the CNS, where it is always better to use at least two drugs with good intracranial diffusion and, when severe, a triple combination (Ambrosioni et al., 2010).During the administration of amikacin and imipenem, doses should be adjusted according to renal function and auditory function should be monitored.In addition, in LTx, co-administration of cyclosporine or tacrolimus with amikacin may enhance the nephrotoxicity of aminoglycosides.(Clark, 2009, Ambrosioni et al., 2010, modified).TMP/SMX, trimethoprim-sulfamethoxazole.

INFECTION
As regards the carbapenems, imipenem and meropenem have a similar spectrum of activity and effectiveness and, although the latter is somewhat less effective against N. asteroides complex (NAC) and more effective against N. brasiliensis and N. otitidiscaviarum, it is preferred in cases with CNS involvement, since imipenem has been associated with

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Lung Diseases -Selected State of the Art Reviews 400 seizures.Ertapenem has slightly less activity against several species of Nocardia and doripenem, with a similar spectrum of antibiotic activity to meropenem (Lai et al., 2009) but without CNS penetration, has not been tested.
Other beta-lactam antibiotics such as the third-generation cephalosporins ceftriaxone or cefotaxime, due to their good CNS penetration, may be combined with other antimicrobials for treatment of intracranial infections with good results, depending on the species of Nocardia, since some may be intrinsically resistant (Garlando et al., 1992;Durmaz et al., 2001).

Other alternatives
The literature includes experiences of varying degrees of success with other antibiotic treatments, including minocycline and tigecycline, macrolides, ampicillin, piperacillin/ tazobactam, fluoroquinolones (ciprofloxacin, moxifloxacin and gatifloxacin), but experiences are limited and there is not enough scientific basis to include them in general recommendations.With moxifloxacin, which is active against N. farcinica (Hanse et al., 2008), good results have been obtained in some cases; however, in others there has been recurrence in the CNS, despite its activity and having achieved high levels of drug in the abscess material (Dahan et al., 2006).
In recent years, the oxazolidinone linezolid has been gathering attention due to its excellent activity against all species of Nocardia, including N. farcinica (Brown-Elliot et al., 2001).It has been used successfully, even in monotherapy, in six patients with disseminated nocardiosis (Moylett et al., 2003) and also in combination with other antimicrobials (Lewis et al., 2003;Rivero et al., 2008).It has extensive intravenous and oral bioavailability, crosses the bloodbrain barrier, does not require renal or liver dose adjustments and has few interactions with immunosuppressive agents (Jodlowski et al., 2007), which makes it a very reasonable alternative as first-or second-line treatment of Nocardia infections in LTx (especially in cases of kidney involvement).Limitations on long-term use are conditioned by its high cost and possible toxicity, since, in addition to its minor adverse effects such as rash, nausea and vomiting, it may also induce, less frequently, thrombocytopaenia, aplastic anaemia, peripheral neuropathy, lactic acidosis and serotonin syndrome (Beekmann et al., 2008).

Duration of treatment
The optimal duration of treatment has not been standardised, but should be several months due to the difficulty of eradicating Nocardia and frequency of relapse (Sorrell et al., 2005).Most patients begin to improve in 1-2 weeks, but more severe cases must follow an additional 3 to 4 weeks of intravenous therapy before switching to oral treatment.Cerebral nocardiosis requires at least 9-12 months of treatment, whereas soft tissue and lung presentations require 6-12 months, depending on the clinical course and response to treatment (Clark, 2009).If the infection is associated with a central venous catheter, this must be removed and followed by administration of antibiotic treatment for several months.

Consolidation therapy
When the patient improves and intravenous treatment can be withdrawn, oral alternatives may include amoxicillin-clavulanate, TMP/SMX, linezolid, clarithromycin, ciprofloxacin and moxifloxacin as consolidation therapy.There are no trials comparing the effects of these antibiotics.Some studies recommend a dual oral combination after severe infections, such as those involving the CNS (Sullivan & Chapman, 2010).
After discontinuation of treatment, the patient should be monitored for a minimum of one year to detect possible relapses.In some hospitals, prophylaxis is maintained for as long as the patient remains immunosuppressed (King et al., 1993;Poonyagariyagorn et al., 2008).

Evolution
Despite combined treatment, the prognosis is variable and depends heavily on: the extent of infection, the underlying conditions of the organ recipients and co-infections that precede or coincide with Nocardia infection (Peleg et al., 2007b).Overall mortality is around 40% (Husain et al., 2002).In CNS infections it is 30-55% (Mamelak et al., 1994) and in the lung about 14-18% (Poonyagariyagorn et al., 2008;Morales et al., 2011).The prognosis is better in isolated skin lesions, in which some studies report up to 90% healing.
Delayed diagnosis and discontinuation of treatment has also been associated with poor prognosis.There is little information on the crude mortality rate, because in many cases the patient died due to multiple causes and it is difficult to determine the impact of Nocardia infection on the final outcome.

Prevention/Prophylaxis
Given the low incidence of Nocardia infection in LTx and that its onset tends to be late, primary chemoprophylaxis is not indicated.However, in patients with solid organ transplant in general, TMP/SMX is administered daily for at least six months posttransplant and then on alternate days indefinitely to prevent infection by Pneumocystis jiroveci (Hewayama et al., 2011).Some works suggest that this prophylaxis reduces infection by Nocardia and this benefit extends to other microorganisms.Nevertheless, there is an increasing number of reports of TMP/SMX susceptible Nocardia isolations in transplant recipients taking this antibiotic as prophylaxis (Roberts et al., 2000;Husain et al., 2002;Poonyagariyagorn et al., 2008;Khan et al., 2008;Minero et al., 2009).This fact raises doubts concerning the prophylactic role of TMP/SMX to prevent nocardiosis.The lack of efficacy may be due to different protocols in SOT recipients.Relapse has also been documented in LTx (Poonyagariyagorn, 2008;Roberts et al., 2000) after one cycle of antibiotic treatment, possibly due to short-term duration or interruption, but the data are controversial with regard to recommending secondary prophylaxis.Long-term prophylaxis to prevent these relapses is only suggested in a few hospitals.
Moreover, considering that Nocardia is a ubiquitous microorganism, it is important to teach LTx to alter their lifestyle and adopt a careful and alert attitude in order to reduce their high epidemiological risk of exposure, especially in the community.Recently, Avery and Michaels have published an excellent guide to strategies for avoiding infection following solid organ transplantation (Avery & Michaels, 2009).

Peri-Transplant considerations 7.1 Pre-Transplant
The future LTx may have had an infection that has already been treated or that is still active during the pre-transplant evaluation period.In fact, this happens with some frequency in patients with cystic fibrosis (CF).Neither situation, according to most experts and to our own experience, is an absolute contraindication for transplantation.Nevertheless, the patient must be monitored closely, being aware of possible relapses, complications or dissemination of infection, and ensuring full compliance with treatment.

Post-Transplant
Considerations are similar when nocardiosis occurs after transplantation, as we have previously discussed in the text, paying special attention to possible relapses.

Donor
Even though there is no documented case of Nocardia infection in the organ donor, nocardiosis would hypothetically be a relative contraindication for transplantation.In any case, treatment would be obligatory.

Final considerations
The practice of organ transplantation in general and lung transplantation in particular is a growing technique in the clinical setting and transplant recipients inevitably need immunosuppressive treatment.Therefore, the appearance of opportunistic infections, specifically nocardiosis, is expected.Given the nonspecific clinical and radiological signs, diagnosis of these infections is based on high clinical suspicion, proper sample collection and rapid microbiological diagnostic methods to confirm it and properly orient the antibiotic treatment as early as possible.The future challenge seems to be the application of molecular methods directly on the sample (Couble et al., 2005) and perhaps fewer but more effective and less toxic antibiotics, less aggressive immunosuppressive therapy and, overall, to try to avoid infection with a healthy lifestyle.

Table 2 .
Antibiotics suggested for Nocardia infections in organ recipients