Background
Penicillium marneffei is capable of causing fatal systemic mycosis in immunocompromised individuals, especially in HIV-positive patients [
1‐
3]. However, the incidence of
Penicillium marneffei infection in both immunocompetent and immunocompromised patients without HIV infection has shown a marked increase in recent years [
3].
Penicillium marneffei infection can cause immune function decline and deficiency in T-cell-mediated immunity even in a healthy host, such as haematological malignancies, colon cancer, myasthenia gravis, mixed connective tissue disease, transplant rejection, systemic lupus erythematosus, diabetes mellitus, and corticosteroids or immunosuppressive agents [
3,
4]. There are numerous retrospective studies investigating the differences of penicilliosis among patients with and without HIV infection [
3‐
8]. However, no studies have observed differences in
Penicillium marneffei infection among HIV-negative patients with and without underlying disease. This retrospective study is the first time to describe differences in clinical features, immune status, treatment, and outcomes and to elucidate the important factors that influence successful treatment and prognosis of HIV-negative patients with penicilliosis with and without underlying disease.
Methods
Patient population
A retrospective study was conducted between January 1, 2003 and August 1, 2014 at the First Affiliated Hospital of Guangxi Medical University. Consecutive patients diagnosed with Penicilliosis were eligible. HIV-negative patients with Penicillium marneffei infection were included and divided into two groups: patients with underlying disease (Group D) and those without underlying disease (Group ND). Patients who were HIV-positive were excluded. The patients’ clinical records were reviewed for basic information, medical history, auxiliary examination results, and treatments, and summarised for analysis.
This study was approved by the Faculty of Medicine, The First Affiliated Hospital of Guangxi Medical University Ethical Committee. All patients provided written informed consent.
Diagnosis criteria for Penicillium marneffei infection
There were two methods used for pathological and pathogen examination. In the first method, cultures of clinical specimens, including blood, sputum, lymph node, lung tissue, bone, and bone marrow, were established on Sabouraud’s dextrose agar at 25 °C and 37 °C. Positive cultures for
Penicillium marneffei were characterised by dimorphic fungi that grew as a mould at 25 °C and as yeast at 37 °C. A unique characteristic of
Penicillium marneffei mould is the presence of a soluble red pigment that diffuses into the agar making the reverse side appear either pink or red at 25 °C [
2].
For the second method, the yeast form of
Penicillium marneffei was identified by cytology and histopathology from tissues and secretions by periodic acid-Schiff staining or Wright’s staining.
Penicillium marneffei has a characteristic morphology, including a transverse septum [
6].
Patients’ sera were tested in duplicate at our hospital and the Guangxi Center for Prevention and Control by using enzyme-linked immunosorbent assay (Enzymun-Test Anti-HIV 1 + 2; Boehringer Mannheim GmbH Diagnostica) and particle agglutination test (Serodia-HIV; Fujirebio Inc., Tokyo, Japan).
Inclusion and exclusion criteria
Inclusion criteria were as follows: i) HIV negative; and ii) a clear diagnosis of Penicillium marneffei infection.
Patients diagnosed with Penicillium marneffei infection but who were HIV-positive were excluded.
Statistical analysis
Clinical data were analysed as percentages (%), means and standard deviations, and medians and interquartile ranges, as appropriate. Comparison of the demographic data and clinical characteristics of HIV-negative patients with and without underlying diseases was performed using the Student’s t-test, Mann–Whitney U test, chi-square test, or Fisher’s exact test, as appropriate. Survival curves were estimated using Kaplan-Meier analyses, and the differences in survival rates between the two groups were compared using the log-rank test. Univariate analysis was performed to assess significant differences in clinical characteristics that influence overall survival. Multivariate analysis was performed using Cox regression analysis for significant variables identified by the univariate analysis. All statistical analyses were performed using the Statistical Package for the Social Sciences (Windows version 16.0; SPSS Inc., Chicago, IL, US). Two-tailed tests were used, with a P-value of < 0.05 indicating statistical significance.
Discussion
Penicilliosis has been reported in immunocompromised patients with HIV infection, but also in patients free of HIV infection. Certain diseases can cause immune function decline and immune disorders, such as haematological malignancies, colon cancer, myasthenia gravis, mixed connective tissue disease, transplant rejection, systemic lupus erythematosus, diabetes mellitus, as well as the use of corticosteroids or immunosuppressive agents, even in HIV-negative [
3,
4]. The incidence of
Penicillium marneffei infection in patients without HIV infection has shown a marked increased in recent years [
3,
5‐
8]. In our study, during an 11-year period,
Penicillium marneffei infection was present in 43 (43/109, 39.45 %) patients without HIV infection. Among these patients, 41.86 % had underlying diseases, with diabetes being the most common; 58.14 % had no underlying diseases, which suggests that
Penicillium marneffei infection is not a rare entity, but is also common in HIV-negative individuals and even healthy hosts.
The common clinical manifestations among HIV-negative patients were similar to those in previous reports [
6]. However, there were no statistically significant differences between Group D and Group ND in clinical manifestations and laboratory findings, with the exception of thoracalgia, which was more common in HIV-negative patients with underlying disease (
P < 0.05).
Previous work has postulated that deficiency of CD4+ T-cell-mediated immunity plays a key pathogenic role in AIDS patients and may also be involved in immunocompromised HIV-negative patients, potentially leading to
Penicillium marneffei infection [
6‐
9]. In our study, CD8 T-cell counts, CD8 T-cell percentages, and serum immunoglobulin levels did not differ between the two groups (
P > 0.05). However, HIV-negative patients without underlying disease had higher lymphocyte cell counts, CD4 cell counts, and CD4 T-cell percentages than those with underlying disease. The mean CD4 cell count was below normal levels in HIV-negative patients with underlying disease. In addition, in the univariate analysis, only underlying disease, CD4 cell percentage, and T lymphocyte cell percentage were significantly associated with overall survival. Thus, HIV-negative patients with underlying disease, such as cancer, mixed connective tissue disease, transplant rejection, systemic lupus erythematosus, diabetes mellitus, and Langerhans cell histiocytosis, and those taking corticosteroids or immunosuppressive agents, exhibited worse CD4 T-cellular immunodeficiency during the course of the disease than HIV-negative patients without underlying disease. Browne et al. and Lee et al. reported that autoantibody to interferon-ɤ is associated with the new clinical syndrome of adult-onset immunodeficiency [
8,
10,
11]. This may be another cause of cell-mediated immunity defects in patients who are not infected with HIV. This also suggests that underlying disease, CD4 cell percentage, and T lymphocyte cell percentage may be potential risk factors affecting prognosis.
Specifically, 32.5 % of patients had osteolytic lesions, and all of these were HIV-negative individuals; no HIV-positive patients exhibited such lesions. Thus, osteolytic lesions are one of the most common and important clinical characteristics of
Penicillium marneffei infection, which can easily be neglected in HIV-negative patients [
12]. Serous effusions are often thought to reflect hypoalbuminaemia, connective tissue disease, or tuberculosis. However, in our study, 81.39 % of patients had multi-exudate effusion, which suggests that exudate effusions are equally common in
Penicillium marneffei infection in HIV-negative individuals. However, the mechanism by which effusion is generated is unclear; the cause may be inflammation initiated by disseminated
Penicillium marneffei infection, or may directly involve the thoracic cavity.
The most common diagnostic method of Penicilliosis was cultures from biopsy tissues and secretions, followed by biopsy pathology, bone marrow cultures, and blood cultures, which is consistent with the presence of skin lesions and lymph node lesions. Histopathology showed not only classic yeast-like fungi in proliferative macrophages, but also granulomas, gaseous necrosis, and small abscesses, suggesting that the immune system can play a defensive role.
There were relatively few cases of
Penicillium marneffei infection among HIV-negative patients with and without underlying disease. The current recommendation for severe disease in HIV-positive patients is intravenous amphotericin B at a dosage of 0.6 mg∙kg
−1∙d
−1 for 2 weeks, followed by oral itraconazole 200 mg twice daily for 10 weeks. For mild disease, the recommendation is oral itraconazole at a dose of 200 mg twice daily for 8–12 weeks [
6].
In this study, we found that HIV-negative patients with underlying disease had a worse prognosis and greater treatment difficulty than those without underlying disease. The reason for this is that HIV-negative patients with underlying disease have more obvious immune deficiency and CD4+ T cell-mediated immune deficiency than patients without underlying disease. Some diseases have the potential to damage immune function, especially CD4 T-cellular immune function. The majority of patients in this study received treatment for Penicillium marneffei infection with intravenous amphotericin B at a dosage of 0.6 mg∙kg−1∙d−1 for 4–8 weeks, followed by oral itraconazole or fluconazole 200 mg twice daily for 6–12 months, even treated for up to 2 years, which was significantly longer than the current recommendation for severe disease in HIV-positive patients. Oral itraconazole at a dosage of 200 mg once daily has been recognised as secondary prophylaxis. Thus, timely, effective, and longer courses of antifungal treatment, combined with support of the immune system, are important to improve patient prognosis. The longest duration for relapse was 4 years in this study; therefore, longer-term secondary prophylaxis and follow-up may be necessary to prevent relapse and improve prognosis. The important factors to assess when to discontinue secondary prophylaxis treatment include lymphocyte cell counts, CD4 cell counts, CD4 T-cell percentages, lung and bone imaging, and negative culture of clinical specimens.
Conclusion
Penicillium marneffei can infect individuals without HIV infection and can cause fatal systemic mycosis. There were no clear differences in clinical manifestations among HIV-negative patients with and without underlying disease. However, Penicillium marneffei in patients without HIV infection, in combination with certain diseases, may cause immune function decline and a deficiency in T-cell-mediated immunity during the course of the disease. Underlying disease, CD4 cell percentage, and T lymphocyte cell percentage may be potential risk factors affecting prognosis. Timely, effective, and longer courses of antifungal treatments, in combination with treatments to improve immune function, are important in improving prognoses.
Consent
Written informed consent was obtained from the patients for publication of this article and any accompanying images. Copies of the written consents are available for review.
Competing interests
The authors declare that they have no conflict of interests.
Authors’ contributions
YQ made substantial contributions to the conception and design of the study; acquisition, analysis, and interpretation of the data; and drafting of the manuscript. JZ and HL made substantial contributions to the conception and design of the study; acquisition, analysis, and interpretation of the data; and critical revision of the manuscript for important intellectual content. JZ and XZ also gave final approval of the version to be published and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy and integrity of any part of the work are appropriately investigated and resolved. XZ and DLgave final approval of the version to be published. YQ and CT participated in analysis and interpretation of the data and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. YQ and JZ conceived of the study, participated in its design, and helped to draft the manuscript. All authors read and approved the final manuscript.