Objectives: Aspergillus has become an increasingly frequent cause of life-threatening opportunistic infections in liver transplant recipients. This study seeks to determine the incidence of invasive aspergillosis in liver transplant recipients using routine and molecular methods in a teaching hospital in Shiraz, the unique center for liver transplant in Iran.
Materials and Methods: Four hundred eight recipients who underwent liver transplant were followed for Aspergillus infections by microscopic examination, culture, and nested polymerase chain reaction. Blood samples were cultured by bedside inoculation to BACTEC medium.
Results: The female-to-male ratio was 151:257 (mean age, 29.6 years; mean hospitalization, 26 days). Sensitivity and specificity of the nested polymerase chain reaction was 92.8% and 94%. Aspergillosis was detected in 19 recipients (4.6%) by routine and molecular method (4 proven, 10 probable, and 5 possible) of whom 12 recipients died (63.2%).
Conclusions: This study found the incidence rate of invasive aspergillosis as an uncommon complication of liver transplant recipient cases but associated with poor outcomes. The rate is consistent with those reported in previous studies, but molecular assay that is more-sensitive and specific was used in the present study.
Key words : Nested-PCR, Fungal infection, A. flavus, A. fumigatus
Invasive aspergillosis occurs to 1% to 8% of liver transplant recipients and is associated with a high mortality rate, ranging from 60% to 80% (1, 2). Aspergillus species usually are prone to dissemination in immunocompromised individuals, and rapidly developing foci may appear in several areas of the body. The process may progress to a life-threatening point if rescue therapy is delayed. After solid-organ transplant, providing a balance between immunosuppression to block the rejection, and prevention of infection is critical (3). Other factors predisposing liver recipients to fungal infections are the extensive use of broad-spectrum antibiotics, delayed oral intake, inadequate nutrition, numerous long-term drains and catheters, prolonged hospitalizations, need for intensive care, organ dysfunctions, coinfections, advanced age, and the need for re-exploration (4, 5). Despite extensive investigation on methods such as serologic techniques to improve the rapid diagnosis of these infections, the diagnosis of invasive mycoses remains largely dependent on clinical presentations.
This study sought to determine the incidence rate of invasive aspergillosis in liver transplant recipients by routine and molecular methods in a teaching hospital in Shiraz, the unique center for liver transplant in Iran.
Materials and Methods
From March 2004 to March 2008, all recipients who underwent liver transplant at the solid-organ transplant unit in Nemazi Hospital, Shiraz University of Medical Sciences, were followed for Aspergillus infections during the first 6 months after transplant. This center is unique in Iran for liver transplant.
Liver transplant recipients received fluconazole (400 mg/day) for 21 days after transplant and also received prednisolone, mycophenolate mofetil, and tacrolimus. Whenever clinicians suspected fungal infections by clinical and/or radiologic signs, clinical samples (ie, urine, cerebrospinal fluid, pleural and abdominal fluids, bronchoalveolar fluid from lavage, biopsy, blood, and sputum) were examined for fungal infections by microscopic examination and culture on Sabouraud dextrose agar (Merck, Darmstadt, Germany). Blood samples were cultured by bedside inoculation to BACTEC medium (Becton-Dickinson, Sparks, MD, USA), and 2 blood clot samples were collected at 1-week intervals for molecular examination.
Sera from the patients were extracted for Aspergillus DNA by QIAamp DNA Minikit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s recommendations. The polymerase chain reaction was performed with 2 sets of primers (nested polymerase chain reaction) according to Yamakami and associates (6). That is based on a comparison of the sequence of 18S rRNA genes of Aspergillus species and can identify all the Aspergillus (A) specious (panfungal aspergillosis).
To determine the sensitivity and to limit the assay for fungal pathogens in the blood, suspensions of serum with Aspergillus (A. flavus and A. fumigatus) conidia (1 to 105 conidia/mL) was diluted, and each solution was cultured for colony count and used for DNA extraction and polymerase chain reaction.
Cases were identified, using the consensus definitions for the proven, probable, and possible infections developed by the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) and Pappas and associates (7, 8). In cases in which the recipients presented with clinical and/or radiologic signs, and microscopic examination or culture of the clinical samples or polymerase chain reaction from serum were positive for Aspergillus species, they were treated with antifungal agents. Recipients were followed for any complications in the hospital, and nested polymerase chain reaction was rechecked after treatment with antifungal agents. Fifty liver transplant recipients with negative clinical and/or radiologic signs of invasive aspergillosis were examined with nested polymerase chain reaction as controls. Statistical analyses were performed with SPSS software for Windows (Statistical Product and Service Solutions, version 15.0, SSPS Inc, Chicago, IL, USA).
The ethics committee of Prof. Alborzi Clinical Microbiology Research Center has reviewed and approved the study, considering the patients written, informed consent before beginning the study. The protocol conforms to the ethical guidelines of the 1975 Helsinki Declaration.
From March 2004 to March 2008, 408 recipients underwent liver transplant. The female-to-male ratio was 151:257 (mean age, 29.6 years; range, 1-62 years). A range of 20 to 29 years was the highest frequency among the recipients; the mean hospitalization was 26 days (range, 9-150 days).
Of the 408 recipients, 209 developed complications, and 43 patients were suspicious to invasive aspergillosis. Clinical samples from them (209 cases) were examined by routine methods (microscopic examination and culture) and nested polymerase chain reaction also was performed on the sera from these patients.
The lower limit of detection of this polymerase chain reaction (sensitivity level) assay was 1 colony forming unit/mL of serum. All the infected recipients except 1 with an eye infection had positive nested polymerase chain reaction results. Of the control liver transplant recipients, 3 who had negative clinical and/or radiologic signs of invasive aspergillosis were positive with nested polymerase chain reaction (false positive). The sensitivity, specificity of this nested polymerase chain reaction in proven, and probable cases was 92.8% and 94%. Aspergillosis was reported in 19 recipients (4.6%) with clinical signs (pneumonitis, invasive sinusitis, central nervous system infection, and systemic infection), consisting of 13 males and 6 females (mean age, 21.7 years; range, 2-57 years). Classification of these 19 cases according to the consensual EORTC/MSG criteria was as follows: 4 proven, 10 probable, and 5 possible. None of the patients with invasive aspergillosis had a positive blood culture result. Table 1 is a summary of the baseline characteristics of the recipients with invasive aspergillosis.
Invasive aspergillosis developed within 2 months of liver transplant (mean, 40 days posttransplant). There were 4 proven cases based on the mycologic criteria for detecting invasive aspergillosis. A. fumigatus was detected in cerebral spinal fluid from 1 recipient and in sinus biopsy from another. A. niger was found in the culture of an eye biopsy of 1 recipient with an eye infection and was also isolated from the abdominal culture of another recipient. As for probable invasive aspergillosis, there were 10 cases. A. fumigatus and A. flavus were isolated from the respiratory tracts of 8 patients, and typical hyphae observed in 2 other patients with pulmonary infections using wet mounting with negative culture. Five recipients (possible) with fever were unresponsive to the antibacterials. Despite antifungal therapy, 12 out of 19 recipients with invasive aspergillosis died (63.2%).
Treatment with antifungal agents were successful, the nested polymerase chain reaction results were negative in the second week after initiation of therapy. In other cases, the results remained positive until death.
Despite conventional therapy with amphotericin B, invasive aspergillosis in liver transplant recipients is associated with poor response rates and a high mortality rate. Successful treatment of established fungal infection requires early diagnosis, aggressive debridement when possible, reduction in immunosuppression, and fungicidal therapy (9, 10). Cultures of opportunistic fungi in certain settings such as Aspergillus in respiratory samples from immunosuppressed patients may be associated with infection. For diagnosis of invasive aspergillosis, serodiagnosis remains investigational, and microbiologic antifungal resistance has been increasingly reported.
Accordingly, rapid diagnosis for starting antifungal therapy is desired. Serum- or plasma-based polymerase chain reaction assays have shown improved specificity without loss of sensitivity. Compared with the culture for A. fumigatus, polymerase chain reaction was 19.4 times more sensitive (11). A sensitivity of 79% to 100%, and a specificity of 81.3% to 93% have been documented, depending on methodology (12-15). Sensitivity and specificity of nested polymerase chain reaction used in the present study were comparable to other studies.
In Fortún and associates (2003), invasive aspergillosis was observed in 13 of the 208 liver transplant recipients (6.25%) (16). In our study, aspergillosis was identified in 19 cases (4.6%), and diagnosis was confirmed when clinical and/or radiologic symptoms were associated with either positive Aspergillus species culture or positive nested polymerase chain reaction, for which antifungal therapy with voriconazole was initiated.
The median time to onset of invasive aspergillosis after transplant was 17 days in 1 study (1) and 14.5 days in another (17). More than half of the Aspergillus infections in liver transplant recipients now occur more than 3 months after transplant (18). In the present study, the median time for incidence of invasive aspergillosis was 40 days. This is related to various factors such as age, parenteral antifungals and antibacterials (within 4 weeks pretransplant or posttransplant), and steroid therapy.
Retransplant of livers account for 10% to 15% of the liver transplants performed (9) that is an important risk factor for invasive aspergillosis. In the present study, only 1 recipient who was infected with Aspergillus was retransplanted. Other risk factors including rejection (early or late), bacterial infections, viral infections (Epstein-Barr virus, hepatitis B surface antigen, Cytomegalovirus) and respiratory history (19) also were evident in our recipients whose characteristics are listed in Table 1. The mortality rate in liver transplant recipients with invasive aspergillosis has been reported to be within the range of 83% to 88% in 1998 to 1999 (20, 21) and declared to be from 40% to 80% in 2002 (9, 1). In the current study, the mortality rate was found to be rather high (63.2%), which might have been due to the delay in both diagnosis and treatment.
This 4-year long study, the first of its kind in Iran, found the incidence rate of invasive aspergillosis as an uncommon complication of liver transplant recipient cases, but it continues to be associated with poor outcomes. This rate is comparable with previous reports, except that molecular assay, which is more-sensitive and more-specific, was used in our study.
Volume : 8
Issue : 3
Pages : 220 - 223
From the 1Prof. Alborzi Clinical Microbiology Research Center, and the
2Department of Organ Transplant Research Center, Shiraz University of Medical
Sciences, Shiraz, Iran
Acknowledgements: This work was supported by the grants from the Prof Alborzi Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran. We would like to thank H. Khajehei, PhD, for providing editorial assistance, and Mrs. Parisa Jonghorban who helped with sampling.
Address reprint requests to: Parisa Badiee, Prof. Alborzi Clinical Microbiology Research Center, Zand Ave, Nemazi Hospital, Post code 7193711351, Shiraz, Iran.
Phone: +98 7116474304
Fax: +98 711 6474303
Table 1. Baseline characteristics of the recipients with invasive aspergillosis.