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Serological surveillance for Penicillium marneffei infection in HIV-infected patients during 2004–2011 in Guangzhou, China
ORIGINAL ARTICLE
MYCOLOGY
Serological surveillance for Penicillium marneffei infection in HIV-infected patients during 2004–2011 in Guangzhou, China Y.-F. Wang1,2, H.-F. Xu3, Z.-G. Han3, L. Zeng1,2, C.-Y. Liang3, X.-J. Chen1,2, Y.-J. Chen1,2, J.-P. Cai4, W. Hao1,2, J. F.-W. Chan4, M. Wang3, N. Fu1,2 and X.-Y. Che1,2 1) Division of Laboratory Medicine, Zhujiang Hospital, 2) Laboratory of Emerging Infectious Diseases, Zhujiang Hospital, Southern Medical University, 3) Guangzhou Centre for Disease Control and Prevention, Guangzhou and 4) State Key Laboratory of Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, Hong Kong Special Administrative Region, China
Abstract Prevalence of disseminated Penicillium marneffei infection is not known in human immunodeficiency virus (HIV)-infected patients. This retrospective study aimed to evaluate the prevalence of and risk factors for disseminated P. marneffei infection in HIV-infected patients during 2004–11 in Guangzhou, China. We tested 8131 archived HIV-infected patient serum samples for P. marneffei-specific mannoprotein (Mp1p) antigen using a highly sensitive and specific ELISA that we previously established. The CD4 count of 2686 cases was determined by flow cytometry. Logistic regression was used to assess predictors of Mp1p antigenaemia. The overall prevalence of disseminated penicilliosis as detected by positive serum Mp1p antigen was 9.36% (761/8131), in good concordance with Platelia™ Aspergillus immunoassay. During 2004–11, the prevalence increased to a peak of 12.58% (158/1256) in 2010 and decreased in 2011. Penicilliosis was strongly associated with progression from HIV to AIDS (OR 4.66, 95% CI 3.94–5.51, p <0.001) and humidity (OR 1.02, 95% CI 1.01–1.03, p 0.002). Disseminated penicilliosis occurred mainly during the rainy seasons (p <0.001). For 2686 cases with known CD4 count, logistic regression showed that CD4 count of <200 cells/μL was a risk factor for penicilliosis (OR 2.90, 95% CI 1.10–7.66, p 0.032), especially when it was <50 cells/μL (OR 24.26, 95% CI 10.63–55.36, p <0.001) during which 28.06% of patients developed disseminated penicilliosis. In conclusion, approximately 9.36% of the HIV-infected patients in our study developed disseminated penicilliosis. Rapid diagnosis may be achieved by performing serological surveillance for Mp1p antigenaemia as a routine procedure for all HIV-infected patients with CD4 count of <50 cells/μL. Clinical Microbiology and Infection © 2014 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved. Keywords: AIDS, HIV, Mp1p, penicilliosis, Penicillium marneffei Original Submission: 21 August 2014; Revised Submission: 4 December 2014; Accepted: 15 December 2014 Editor: E. Roilides Article published online: XXX
Corresponding author: N. Fu, Division of Laboratory Medicine, Laboratory of Emerging Infectious Diseases, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China Corresponding author: M. Wang, Guangzhou Centre for Disease Control and Prevention, Guangzhou, 510440, China Corresponding author: J.F.-W. Chan, Carol Yu Centre for Infection, Department of Microbiology, The University of Hong Kong, Hong Kong Special Administrative Region, China E-mails: [email protected] (N. Fu) [email protected] (M. Wang) [email protected] (J.F.-W. Chan)
Deceased. Prof. X.-Y. Che paid great efforts to conceive and design the study, and edit the article Y.-F. Wang and H.-F. Xu contributed equally to this work.
Introduction Penicillium marneffei is an emerging fungus endemic to South East Asian regions, including southern China, Thailand and Vietnam [1]. Penicilliosis, a systemic disease caused by P. marneffei, mainly occurs in immunocompromised patients, particularly those with AIDS and other forms of cell-mediated immunodeficiencies [2–5]. Penicilliosis is classified as an AIDS-defining illness [6], and its prevalence has increased in parallel with that of human immunodeficiency virus (HIV)
Clin Microbiol Infect 2015; -: 1–6 Clinical Microbiology and Infection © 2014 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved http://dx.doi.org/10.1016/j.cmi.2014.12.014
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infection in recent years [1]. Different prevalences of penicilliosis in AIDS patients requiring hospitalization were reported, varying from 4.4% in Vietnam to 14% in Hong Kong [7,8], but it has not been reported in mainland China, especially for HIV-infected patients, including both outpatients and inpatients. Factors that affect the prevalence include environmental predictors, seasonality and changes in CD4 count [7,9]. HIV-infected patients who have a CD4 count of <200 cells/μL are at a particularly high risk for developing the disease [10]. However, the prevalence and characteristics of penicilliosis in HIV-infected patients, especially in Chinese patients with different CD4 counts, is unknown. The relationship between the prevalence of and common risk factors for penicilliosis in HIV infection remains unclear. Previously, we demonstrated that a cell-wall mannoprotein, Mp1p, could be a potential biomarker for the serodiagnosis of penicilliosis [11–13]. Using this protein, we established a double-antibody sandwich ELISA for Mp1p antigen with high sensitivity and specificity [14]. In this study, we used this immunoassay to assess the prevalence of penicilliosis among HIV-infected patients in Guangzhou during 2004–11. The correlation between the CD4 count of our HIVinfected patients and the seasonality with Mp1p antigen-positive rate was also analysed.
Methods Study design A retrospective study was anonymously conducted to determine the prevalence of penicilliosis in HIV-infected patients. We analysed the data of 8131 archived serum samples of HIVinfected patients in Guangzhou from the Guangzhou Centre for Disease Control and Prevention (GZCDC). These samples were collected from 7734 patients over an 8-year period, and there was at most only one sample for every patient in each year. All patients admitted with HIV or AIDS from 1 January 2004 to 31 December 2011 with demographic information, including sex, age and disease stage (HIV infection or AIDS) were included. HIV-1 infection was identified by routine bloodscreening procedures and confirmed by Western blot analysis. AIDS was defined as positive HIV-1 serology with a CD4 count <200 cells/μL. The archived sera were investigated for Mp1p antigen by sandwich ELISA [14]. In the absence of reference standard results for penicilliosis in these archived sera, we confirmed Mp1p antigen-positive sera by Platelia™ Aspergillus enzyme immunoassay (Bio-Rad Laboratories, Redmond, WA, USA), a commercial kit for galactomannan (GM), also positive for penicilliosis [15], and calculated the concordance rate between Mp1p and GM. Patients with disseminated penicilliosis were defined as having an Mp1p antigen-positive serum, which
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indicated active infection with antigenaemia. Only 2686 out of 8131 cases had available CD4 counts. The average temperature and relative humidity for every month in Guangzhou were collected by the Guangdong Meteorological Service. The study was approved by the Ethics Committee of the Zhujiang Hospital of Southern Medical University. HIV-1 test and CD4 cell count Specimens were screened for the HIV antibody by using two ELISA kits (BioMérieux, Boxtel, the Netherlands; Beijing BGIGBI Biotech, Beijing, China). If either of the ELISAs was positive, a Western blot test was conducted for confirmation (MP Biomedicals Asia Pacific Pte Ltd, Singapore). Whole blood was collected from HIV-infected patients into vials containing EDTA. CD4 counts were analysed by flow cytometry (FACS Calibur™, BD, Franklin Lakes, NJ, USA) as previously described [16]. Double-antibody sandwich ELISA for Mp1p antigen The procedure for the antigen capture ELISA was conducted as described previously with small modifications [14]. Briefly, aliquots of 100 μL/well of undiluted human sera were added to microwell plates (Costar, Corning, NY, USA) coated with capture antibodies and incubated at 37°C for 1 h. After the plates were washed, biotinylated antibody (100 μL/well) was added and incubated for 30 min at 26°C. Following incubation with streptavidin–horseradish peroxidase (Sigma-Aldrich, St Louis, MO, USA), tetramethylbenzidine substrate (KPL, Gaithersburg, MD, USA) was added. The reaction was stopped after 10 min by the addition of 0.3 M sulphuric acid, and the plates were then examined in an ELISA plate reader (Bio-Tek, Winooski, VT, USA) at 450 nm. Detection of GM Galactomannan was detected in Mp1p antigenaemia sera using a Platelia Aspergillus assay, according to the manufacturer’s instructions. Because there was no cut-off value for GM levels in the diagnosis of penicilliosis, we analysed the results using three different GM indices as a cut-off: 0.5, 1.0 and 1.5, as suggested by instructions for other invasive fungal infections. Statistical analysis Instances of Mp1p antigenaemia in different months and years were compared by χ2 test, and a two-sided p value of <0.05 was considered significant. The CD4 count was stratified into five groups (0–50, 50–100, 100–200, 200–500 and >500 cells/μL). Prevalences of disseminated penicilliosis (Mp1p antigen-positive rate) among different groups were compared with χ2 test. A two-sided p value of <0.05 was considered significant. Logistic regression was used to assess predictors of Mp1p antigenaemia
Clinical Microbiology and Infection © 2014 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved, CMI, xxx, 1–6
Please cite this article in press as: Wang Y-F, et al., Serological surveillance for Penicillium marneffei infection in HIV-infected patients during 2004–2011 in Guangzhou, China, Clinical Microbiology and Infection (2015), http://dx.doi.org/10.1016/j.cmi.2014.12.014
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based on several covariates, including sex, age, patient serostatus with HIV/AIDS disease stage or CD4 counts, ambient humidity and temperature. A value of p <0.05 was considered significant. Odds ratios were calculated, and were considered as a risk predictor when OR >1.0. All statistical analyses were performed using SPSS (version 11.5).
Results Laboratory features of patients During 2004–11 in Guangzhou, a total of 8131 HIV-infected patient sera were tested. The patients were predominantly male (male:female = 7.1:2.9), and the median age was 35 years (interquartile range (IQR) 29–43 years). Overall, their HIV infection was mainly sexually acquired (55.06%), but the route of acquisition changed throughout the years. Before 2007, more than 44.52% of HIV infections were acquired via blood product transfusion, whereas more than 54.05% after 2007 were sexually acquired. Overall, 53.98% (4389/8131) of all the HIV-infected patients were in the HIV stage and 46.02% (3742/8131) had AIDS. Prevalence of penicilliosis in HIV-infected patients diagnosed by Mp1p antigenaemia test Mp1p antigenaemia may be found in the active phase of acute or reactivated P. marneffei infection. The number of cases of penicilliosis increased significantly throughout the study period, in parallel with that of HIV infection (Fig. 1a). During the 8 years of the study, the average prevalence of Mp1p antigenaemia was 9.36% (761/8131). It increased from 5.17% in 2004 to a peak of 12.58% in 2010, but decreased to 8.25% in 2011, which was still higher than in 2004 (Table 1). There was significant difference in the prevalence (p <0.001) during the different years of the study period, and the trend of prevalence was strongly associated with the progression of HIV infection to AIDS as shown in Fig. 1(b) (OR 4.66, 95% CI 3.94–5.51, p <0.001). GM detection in Mp1p antigenaemia Galactomannan was detected in 513 Mp1p antigenaemia serum samples, to confirm penicilliosis by Platelia Aspergillus kit. The coincidence between Mp1p and GM was 86.55% (444/513), 73.88% (379/513) or 66.67% (342/513), when an index of 0.5, 1.0 or 1.5 is considered as positive, according to the instructions of the kit. The median GM index is 2.58 (IQR 0.88–6.21). Prevalence of penicilliosis in patients with different CD4 counts Of 280 patients with Mp1p antigenaemia and 2406 without Mp1p antigenaemia, the median CD4 counts were 15 cells/μL
FIG. 1. (a) Number of cases of penicilliosis diagnosed by Mp1p antigenaemia and human immunodeficiency virus (HIV) admissions in Guangzhou during 2004–11. (b) Prevalence of penicilliosis diagnosed by Mp1p antigenaemia in HIV-infected patients and proportions of different stages of AIDS in different years.
(IQR 6–39 cells/μL) and 236.5 cells/μL (IQR 58–396 cells/μL), respectively. Overall, 86.78% (243/280) and 78.57% (220/280) of patients with penicilliosis had CD4 count of <100 cells/μL and 50 cells/μL, respectively. Among those with a CD4 count of TABLE 1. Positive rates of Mp1p antigen in HIV-infected patients from Guangzhou during 2004–2011 Year
No. of samples
Mp1p antigen positive % (No. of samples)
2004 2005 2006 2007 2008 2009 2010 2011 Total
271 527 884 894 1221 1284 1256 1794 8131
5.17 6.45 8.37 9.62 9.42 10.28 12.58 8.25 9.36
(14) (34) (74) (86) (115) (132) (158) (148) (761)
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Please cite this article in press as: Wang Y-F, et al., Serological surveillance for Penicillium marneffei infection in HIV-infected patients during 2004–2011 in Guangzhou, China, Clinical Microbiology and Infection (2015), http://dx.doi.org/10.1016/j.cmi.2014.12.014
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<50 cells/μL, Mp1p antigenaemia accounted for 28.06% (220/ 784), which is much higher than the rate of 1.65% (6/363) in patients with a CD4 count of >500 cells/μL (p <0.001). The results are shown in Table 2. Among the 2686 patients with known CD4 count, logistic regression analysis showed that a CD4 count of <200 cells/μL was associated with penicilliosis (OR 2.90, 95% CI 1.10–7.66, p 0.032), and especially when it was <50 cells/μL (OR 24.26, 95% CI 10.63–55.36, p <0.001). The relationship between relative humidity or temperature and penicilliosis in HIV-infected patients in Guangzhou The average relative humidity and temperature were collected for each month of the year from the Guangdong Meteorological Service. Mp1p antigenaemia rate for each month was calculated. The highest rate of 13.84% was in April (Fig. 2) during the rainy seasons in Guangzhou. There were significant variations in the rates among the 12 months of the year (p <0.001). The prevalence was higher during February to July than it was during the other months, and it coincided with the changes in average relative humidity. Logistic regression showed that Mp1p antigenaemia was related with humidity (OR 1.02, 95% CI 1.01–1.03, p 0.002) but not temperature (OR 1.01, 95% CI 0.99–1.02, p 0.455).
Discussion Penicilliosis is one of the most common opportunistic fungal infections in HIV-infected patients in South East Asian regions, including southern China [1]. The prevalence continues to increase in parallel with the increasing number of cases of HIV infection in these areas [1]. Cases of penicilliosis have also been reported among visitors to South East Asia from countries outside the region [17]. Lack of early and accurate diagnostic tools leads to delayed treatment, which is associated with poor clinical outcome. We previously developed a double sandwich
TABLE 2. Prevalence of Mp1p antigenaemia in HIV-infected patient sera with different levels of CD4 count CD4 count (cell/μL)
Mp1p antigen positive group (% no. of samples)
Mp1p antigen negative group (% no. of samples)
Total (no. of samples)
500 200 ~ 500 100 ~ 200 50~100 < 50 Total
1.65 1.69 4.53 10.36 28.06 10.42
98.35 98.31 95.47 89.63 71.94 89.58
363 1008 309 222 784 2686
(6) (17) (14) (23) (220) (280)
(357) (991) (295) (199) (564) (2406)
FIG. 2. Average prevalence of penicilliosis with Mp1p antigenaemia in human immunodeficiency virus (HIV) -infected patients in different months from 2004 to 2011.
ELISA for Mp1p antigen with high sensitivity and specificity [14]. A modified method for detection in undiluted sera was used in this study to enhance sensitivity, after non-specific binding was excluded by preliminary tests. The specificity was found to be 98.81% (579/586) in 563 healthy human sera, and 23 bloodculture-positive sera (14 samples from 14 candidiasis patients, six samples from one fusaridiosis patient, three samples from three cryptococcosis patients). The sensitivity was 100% (7/7) among seven samples from six penicilliosis patients. There is no significant difference in detection performance between the previous study [14] and this modified method. This work determines the prevalence of active penicilliosis in Guangzhou and evaluates risk factors for penicilliosis in HIV-infected patients. Mp1p antigenaemia is usually found in patients with an active infection, which can be acute or reactivated. The prevalence of active and often disseminated disease is evaluated by the Mp1p antigenaemia test. Among HIV-infected patient serum samples collected during 2004–11, the average prevalence of active penicilliosis was 9.36% (761/8131). This result was different from Thuy Le’s report [7], in which prevalence was approximately 4.4% in AIDS patients in Vietnam. This discrepancy may be related to the different demographics among HIV populations and their exposure to P. marneffei. Another possible reason may be that our immunoassays are more sensitive than blood culture and other diagnostic methods. Mp1p antigenaemia was further confirmed by detection of GM with the Platelia Aspergillus kit. A good concordance rate between Mp1p and GM was observed, which is similar to that reported in Taiwan [15]. But we could not exclude other possible reasons for cross-reactivity in the GM assay, especially the use of piperacillin-tazobactam, amoxicillin-clavulanate [18,19], in HIV-infected patients with opportunistic infections. The prevalence of histoplasmosis, which causes a positive GM
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Please cite this article in press as: Wang Y-F, et al., Serological surveillance for Penicillium marneffei infection in HIV-infected patients during 2004–2011 in Guangzhou, China, Clinical Microbiology and Infection (2015), http://dx.doi.org/10.1016/j.cmi.2014.12.014
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result [20], are relatively low in Guangdong, China [21]. Moreover, aspergillosis is rare in HIV-infected people [22]. As for cryptococcosis. which also may give rise to a positive GM result, we found that cryptococcosis patient sera were negative when tested by our Mp1p antigen assay. The trend of active penicilliosis over the 8 years in the study period was shown in Table 1. The prevalence increased to a peak in 2010 before decreasing in 2011. This behaviour may be related to HIV populations with varying disease stages. The increase of active penicilliosis was positively correlated with the proportion of HIV-infected patients at the AIDS stage, and negatively correlated with the proportion at the HIV infection stage. This result indicates that the prevalence of active penicilliosis is associated with CD4 count. HIV-infected patients with CD4 count <50 cells/μL are prone to developing coinfections with various opportunistic fungal infections. In this study, we found that the median CD4 count of patients with penicilliosis was 15 cells/μL (IQR 6–39 cells/μL), among whom 78.57% had a CD4 count of <50 cells/μL. Mp1p antigenaemia usually occurred when the CD4 count was <50 cells/μL. We stratified the CD4 count into five levels, and calculated the prevalence of penicilliosis in each level. Among those with a CD4 count of <50 cells/μL, 28.06% developed penicilliosis. In addition to the CD4 count, seasonality was reported to be associated with the prevalence of penicilliosis [7,9]. In Guangzhou, warm and humid weather is especially suitable for the growth of fungi. In this study, we studied the relationship among the Mp1p antigen, humidity and temperature. Our results show that the prevalence of active penicilliosis is associated with humidity, but not temperature. These results are in accord with those from a previous study [9]. Besides Mp1p antigen, Mp1p antibody should be detected for diagnosis of penicilliosis. We tried to determine this in all 8131 samples by double antigen sandwich ELISA [23], but obtained a low detection rate (data was not shown). Our considerations or desynchrony of antigen and antibody detection are as follows: (i) the different phases for appearance of antigen and antibody; (ii) an overwhelmingly high amount of antigen may consume most antibodies which influences antibody detection by immunological method; (iii) some patients may be not able to mount an adequate antibody response due to immunosuppression. Therefore, we would redesign the dynamics observation to characterize Mp1p antibody in penicilliosis. Our study has some limitations. First, few consecutive serum samples and CD4 count data were available, which prevented us from monitoring the temporal changes in Mp1p antigen, and their relationships with CD4 count. Second, specimens were collected from the outpatient clinics of GZCDC, where the clinical details including other opportunistic infections, antifungal treatments and prophylaxis might be incomplete. Third,
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in a retrospective study, reference standards such as those for culture, histology and cytology were hard to use to confirm Mp1p antigenaemia results. However, a diagnostic Platelia Aspergillus kit was used, and there was good accordance between the GM and Mp1p antigenaemia tests. Fourth, we did not differentiate penicilliosis from histoplasmosis, although there is very low prevalence of histoplasmosis in China [21]. In conclusion, the prevalence of penicilliosis was approximately 9.36% in Guangzhou, China during 2004–11. Most cases of penicilliosis occurred in patients with CD4 count of <50 cells/μL during the rainy season. As an important opportunistic infection in HIV-infected patients in Guangzhou, increased clinical vigilance for the diagnosis of penicilliosis is needed. Penicilliosis can be fatal without timely and appropriate treatment, especially in AIDS patients. Our immunoassay was high throughput and easy to perform, and would serve as a good serodiagnostic tool for penicilliosis in HIV-infected patients. Serum Mp1p antigen screening in HIV-infected patients, especially in those with CD4 count of <50 cells/μL, may prevent mortality associated with penicilliosis.
Transparency declaration The authors declare no conflicts of interest.
Acknowledgements The work is original and has been partially presented at the 23rd European Congress of Clinical Microbiology and Infectious Diseases (Berlin, Germany, 27–30 April 2013). This work was supported by grants from the National Natural Science Foundation of China (NSFC, 81101299), the Science and Technology Programme of Guangzhou, China (2012Y2-00021), the Project for Engineering Technology Research and Development Centre of Guangdong Province Universities and Colleges (GCZX-A1106), and the Health and Medical Research Fund, Hong Kong Special Administrative Region, China (13121342). We thank Professor K.-Y. Yuen for revising the manuscript. The authors also thank the staff and graduate students of Guangzhou Centre for Disease Control and Prevention and our laboratory for assistance with this study.
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Please cite this article in press as: Wang Y-F, et al., Serological surveillance for Penicillium marneffei infection in HIV-infected patients during 2004–2011 in Guangzhou, China, Clinical Microbiology and Infection (2015), http://dx.doi.org/10.1016/j.cmi.2014.12.014
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Serological surveillance for Penicillium marneffei infection in HIV-infected patients during 2004–2011 in Guangzhou, China Y.-F. Wang1,2, H.-F. Xu3, Z.-G. Han3, L. Zeng1,2, C.-Y. Liang3, X.-J. Chen1,2, Y.-J. Chen1,2, J.-P. Cai4, W. Hao1,2, J. F.-W. Chan4, M. Wang3, N. Fu1,2 and X.-Y. Che1,2 1) Division of Laboratory Medicine, Zhujiang Hospital, 2) Laboratory of Emerging Infectious Diseases, Zhujiang Hospital, Southern Medical University, 3) Guangzhou Centre for Disease Control and Prevention, Guangzhou and 4) State Key Laboratory of Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, Hong Kong Special Administrative Region, China
Abstract Prevalence of disseminated Penicillium marneffei infection is not known in human immunodeficiency virus (HIV)-infected patients. This retrospective study aimed to evaluate the prevalence of and risk factors for disseminated P. marneffei infection in HIV-infected patients during 2004–11 in Guangzhou, China. We tested 8131 archived HIV-infected patient serum samples for P. marneffei-specific mannoprotein (Mp1p) antigen using a highly sensitive and specific ELISA that we previously established. The CD4 count of 2686 cases was determined by flow cytometry. Logistic regression was used to assess predictors of Mp1p antigenaemia. The overall prevalence of disseminated penicilliosis as detected by positive serum Mp1p antigen was 9.36% (761/8131), in good concordance with Platelia™ Aspergillus immunoassay. During 2004–11, the prevalence increased to a peak of 12.58% (158/1256) in 2010 and decreased in 2011. Penicilliosis was strongly associated with progression from HIV to AIDS (OR 4.66, 95% CI 3.94–5.51, p <0.001) and humidity (OR 1.02, 95% CI 1.01–1.03, p 0.002). Disseminated penicilliosis occurred mainly during the rainy seasons (p <0.001). For 2686 cases with known CD4 count, logistic regression showed that CD4 count of <200 cells/μL was a risk factor for penicilliosis (OR 2.90, 95% CI 1.10–7.66, p 0.032), especially when it was <50 cells/μL (OR 24.26, 95% CI 10.63–55.36, p <0.001) during which 28.06% of patients developed disseminated penicilliosis. In conclusion, approximately 9.36% of the HIV-infected patients in our study developed disseminated penicilliosis. Rapid diagnosis may be achieved by performing serological surveillance for Mp1p antigenaemia as a routine procedure for all HIV-infected patients with CD4 count of <50 cells/μL. Clinical Microbiology and Infection © 2014 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved. Keywords: AIDS, HIV, Mp1p, penicilliosis, Penicillium marneffei Original Submission: 21 August 2014; Revised Submission: 4 December 2014; Accepted: 15 December 2014 Editor: E. Roilides Article published online: XXX
Corresponding author: N. Fu, Division of Laboratory Medicine, Laboratory of Emerging Infectious Diseases, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China Corresponding author: M. Wang, Guangzhou Centre for Disease Control and Prevention, Guangzhou, 510440, China Corresponding author: J.F.-W. Chan, Carol Yu Centre for Infection, Department of Microbiology, The University of Hong Kong, Hong Kong Special Administrative Region, China E-mails: [email protected] (N. Fu) [email protected] (M. Wang) [email protected] (J.F.-W. Chan)
Deceased. Prof. X.-Y. Che paid great efforts to conceive and design the study, and edit the article Y.-F. Wang and H.-F. Xu contributed equally to this work.
Introduction Penicillium marneffei is an emerging fungus endemic to South East Asian regions, including southern China, Thailand and Vietnam [1]. Penicilliosis, a systemic disease caused by P. marneffei, mainly occurs in immunocompromised patients, particularly those with AIDS and other forms of cell-mediated immunodeficiencies [2–5]. Penicilliosis is classified as an AIDS-defining illness [6], and its prevalence has increased in parallel with that of human immunodeficiency virus (HIV)
Clin Microbiol Infect 2015; -: 1–6 Clinical Microbiology and Infection © 2014 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved http://dx.doi.org/10.1016/j.cmi.2014.12.014
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infection in recent years [1]. Different prevalences of penicilliosis in AIDS patients requiring hospitalization were reported, varying from 4.4% in Vietnam to 14% in Hong Kong [7,8], but it has not been reported in mainland China, especially for HIV-infected patients, including both outpatients and inpatients. Factors that affect the prevalence include environmental predictors, seasonality and changes in CD4 count [7,9]. HIV-infected patients who have a CD4 count of <200 cells/μL are at a particularly high risk for developing the disease [10]. However, the prevalence and characteristics of penicilliosis in HIV-infected patients, especially in Chinese patients with different CD4 counts, is unknown. The relationship between the prevalence of and common risk factors for penicilliosis in HIV infection remains unclear. Previously, we demonstrated that a cell-wall mannoprotein, Mp1p, could be a potential biomarker for the serodiagnosis of penicilliosis [11–13]. Using this protein, we established a double-antibody sandwich ELISA for Mp1p antigen with high sensitivity and specificity [14]. In this study, we used this immunoassay to assess the prevalence of penicilliosis among HIV-infected patients in Guangzhou during 2004–11. The correlation between the CD4 count of our HIVinfected patients and the seasonality with Mp1p antigen-positive rate was also analysed.
Methods Study design A retrospective study was anonymously conducted to determine the prevalence of penicilliosis in HIV-infected patients. We analysed the data of 8131 archived serum samples of HIVinfected patients in Guangzhou from the Guangzhou Centre for Disease Control and Prevention (GZCDC). These samples were collected from 7734 patients over an 8-year period, and there was at most only one sample for every patient in each year. All patients admitted with HIV or AIDS from 1 January 2004 to 31 December 2011 with demographic information, including sex, age and disease stage (HIV infection or AIDS) were included. HIV-1 infection was identified by routine bloodscreening procedures and confirmed by Western blot analysis. AIDS was defined as positive HIV-1 serology with a CD4 count <200 cells/μL. The archived sera were investigated for Mp1p antigen by sandwich ELISA [14]. In the absence of reference standard results for penicilliosis in these archived sera, we confirmed Mp1p antigen-positive sera by Platelia™ Aspergillus enzyme immunoassay (Bio-Rad Laboratories, Redmond, WA, USA), a commercial kit for galactomannan (GM), also positive for penicilliosis [15], and calculated the concordance rate between Mp1p and GM. Patients with disseminated penicilliosis were defined as having an Mp1p antigen-positive serum, which
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indicated active infection with antigenaemia. Only 2686 out of 8131 cases had available CD4 counts. The average temperature and relative humidity for every month in Guangzhou were collected by the Guangdong Meteorological Service. The study was approved by the Ethics Committee of the Zhujiang Hospital of Southern Medical University. HIV-1 test and CD4 cell count Specimens were screened for the HIV antibody by using two ELISA kits (BioMérieux, Boxtel, the Netherlands; Beijing BGIGBI Biotech, Beijing, China). If either of the ELISAs was positive, a Western blot test was conducted for confirmation (MP Biomedicals Asia Pacific Pte Ltd, Singapore). Whole blood was collected from HIV-infected patients into vials containing EDTA. CD4 counts were analysed by flow cytometry (FACS Calibur™, BD, Franklin Lakes, NJ, USA) as previously described [16]. Double-antibody sandwich ELISA for Mp1p antigen The procedure for the antigen capture ELISA was conducted as described previously with small modifications [14]. Briefly, aliquots of 100 μL/well of undiluted human sera were added to microwell plates (Costar, Corning, NY, USA) coated with capture antibodies and incubated at 37°C for 1 h. After the plates were washed, biotinylated antibody (100 μL/well) was added and incubated for 30 min at 26°C. Following incubation with streptavidin–horseradish peroxidase (Sigma-Aldrich, St Louis, MO, USA), tetramethylbenzidine substrate (KPL, Gaithersburg, MD, USA) was added. The reaction was stopped after 10 min by the addition of 0.3 M sulphuric acid, and the plates were then examined in an ELISA plate reader (Bio-Tek, Winooski, VT, USA) at 450 nm. Detection of GM Galactomannan was detected in Mp1p antigenaemia sera using a Platelia Aspergillus assay, according to the manufacturer’s instructions. Because there was no cut-off value for GM levels in the diagnosis of penicilliosis, we analysed the results using three different GM indices as a cut-off: 0.5, 1.0 and 1.5, as suggested by instructions for other invasive fungal infections. Statistical analysis Instances of Mp1p antigenaemia in different months and years were compared by χ2 test, and a two-sided p value of <0.05 was considered significant. The CD4 count was stratified into five groups (0–50, 50–100, 100–200, 200–500 and >500 cells/μL). Prevalences of disseminated penicilliosis (Mp1p antigen-positive rate) among different groups were compared with χ2 test. A two-sided p value of <0.05 was considered significant. Logistic regression was used to assess predictors of Mp1p antigenaemia
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Please cite this article in press as: Wang Y-F, et al., Serological surveillance for Penicillium marneffei infection in HIV-infected patients during 2004–2011 in Guangzhou, China, Clinical Microbiology and Infection (2015), http://dx.doi.org/10.1016/j.cmi.2014.12.014
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based on several covariates, including sex, age, patient serostatus with HIV/AIDS disease stage or CD4 counts, ambient humidity and temperature. A value of p <0.05 was considered significant. Odds ratios were calculated, and were considered as a risk predictor when OR >1.0. All statistical analyses were performed using SPSS (version 11.5).
Results Laboratory features of patients During 2004–11 in Guangzhou, a total of 8131 HIV-infected patient sera were tested. The patients were predominantly male (male:female = 7.1:2.9), and the median age was 35 years (interquartile range (IQR) 29–43 years). Overall, their HIV infection was mainly sexually acquired (55.06%), but the route of acquisition changed throughout the years. Before 2007, more than 44.52% of HIV infections were acquired via blood product transfusion, whereas more than 54.05% after 2007 were sexually acquired. Overall, 53.98% (4389/8131) of all the HIV-infected patients were in the HIV stage and 46.02% (3742/8131) had AIDS. Prevalence of penicilliosis in HIV-infected patients diagnosed by Mp1p antigenaemia test Mp1p antigenaemia may be found in the active phase of acute or reactivated P. marneffei infection. The number of cases of penicilliosis increased significantly throughout the study period, in parallel with that of HIV infection (Fig. 1a). During the 8 years of the study, the average prevalence of Mp1p antigenaemia was 9.36% (761/8131). It increased from 5.17% in 2004 to a peak of 12.58% in 2010, but decreased to 8.25% in 2011, which was still higher than in 2004 (Table 1). There was significant difference in the prevalence (p <0.001) during the different years of the study period, and the trend of prevalence was strongly associated with the progression of HIV infection to AIDS as shown in Fig. 1(b) (OR 4.66, 95% CI 3.94–5.51, p <0.001). GM detection in Mp1p antigenaemia Galactomannan was detected in 513 Mp1p antigenaemia serum samples, to confirm penicilliosis by Platelia Aspergillus kit. The coincidence between Mp1p and GM was 86.55% (444/513), 73.88% (379/513) or 66.67% (342/513), when an index of 0.5, 1.0 or 1.5 is considered as positive, according to the instructions of the kit. The median GM index is 2.58 (IQR 0.88–6.21). Prevalence of penicilliosis in patients with different CD4 counts Of 280 patients with Mp1p antigenaemia and 2406 without Mp1p antigenaemia, the median CD4 counts were 15 cells/μL
FIG. 1. (a) Number of cases of penicilliosis diagnosed by Mp1p antigenaemia and human immunodeficiency virus (HIV) admissions in Guangzhou during 2004–11. (b) Prevalence of penicilliosis diagnosed by Mp1p antigenaemia in HIV-infected patients and proportions of different stages of AIDS in different years.
(IQR 6–39 cells/μL) and 236.5 cells/μL (IQR 58–396 cells/μL), respectively. Overall, 86.78% (243/280) and 78.57% (220/280) of patients with penicilliosis had CD4 count of <100 cells/μL and 50 cells/μL, respectively. Among those with a CD4 count of TABLE 1. Positive rates of Mp1p antigen in HIV-infected patients from Guangzhou during 2004–2011 Year
No. of samples
Mp1p antigen positive % (No. of samples)
2004 2005 2006 2007 2008 2009 2010 2011 Total
271 527 884 894 1221 1284 1256 1794 8131
5.17 6.45 8.37 9.62 9.42 10.28 12.58 8.25 9.36
(14) (34) (74) (86) (115) (132) (158) (148) (761)
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Please cite this article in press as: Wang Y-F, et al., Serological surveillance for Penicillium marneffei infection in HIV-infected patients during 2004–2011 in Guangzhou, China, Clinical Microbiology and Infection (2015), http://dx.doi.org/10.1016/j.cmi.2014.12.014
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<50 cells/μL, Mp1p antigenaemia accounted for 28.06% (220/ 784), which is much higher than the rate of 1.65% (6/363) in patients with a CD4 count of >500 cells/μL (p <0.001). The results are shown in Table 2. Among the 2686 patients with known CD4 count, logistic regression analysis showed that a CD4 count of <200 cells/μL was associated with penicilliosis (OR 2.90, 95% CI 1.10–7.66, p 0.032), and especially when it was <50 cells/μL (OR 24.26, 95% CI 10.63–55.36, p <0.001). The relationship between relative humidity or temperature and penicilliosis in HIV-infected patients in Guangzhou The average relative humidity and temperature were collected for each month of the year from the Guangdong Meteorological Service. Mp1p antigenaemia rate for each month was calculated. The highest rate of 13.84% was in April (Fig. 2) during the rainy seasons in Guangzhou. There were significant variations in the rates among the 12 months of the year (p <0.001). The prevalence was higher during February to July than it was during the other months, and it coincided with the changes in average relative humidity. Logistic regression showed that Mp1p antigenaemia was related with humidity (OR 1.02, 95% CI 1.01–1.03, p 0.002) but not temperature (OR 1.01, 95% CI 0.99–1.02, p 0.455).
Discussion Penicilliosis is one of the most common opportunistic fungal infections in HIV-infected patients in South East Asian regions, including southern China [1]. The prevalence continues to increase in parallel with the increasing number of cases of HIV infection in these areas [1]. Cases of penicilliosis have also been reported among visitors to South East Asia from countries outside the region [17]. Lack of early and accurate diagnostic tools leads to delayed treatment, which is associated with poor clinical outcome. We previously developed a double sandwich
TABLE 2. Prevalence of Mp1p antigenaemia in HIV-infected patient sera with different levels of CD4 count CD4 count (cell/μL)
Mp1p antigen positive group (% no. of samples)
Mp1p antigen negative group (% no. of samples)
Total (no. of samples)
500 200 ~ 500 100 ~ 200 50~100 < 50 Total
1.65 1.69 4.53 10.36 28.06 10.42
98.35 98.31 95.47 89.63 71.94 89.58
363 1008 309 222 784 2686
(6) (17) (14) (23) (220) (280)
(357) (991) (295) (199) (564) (2406)
FIG. 2. Average prevalence of penicilliosis with Mp1p antigenaemia in human immunodeficiency virus (HIV) -infected patients in different months from 2004 to 2011.
ELISA for Mp1p antigen with high sensitivity and specificity [14]. A modified method for detection in undiluted sera was used in this study to enhance sensitivity, after non-specific binding was excluded by preliminary tests. The specificity was found to be 98.81% (579/586) in 563 healthy human sera, and 23 bloodculture-positive sera (14 samples from 14 candidiasis patients, six samples from one fusaridiosis patient, three samples from three cryptococcosis patients). The sensitivity was 100% (7/7) among seven samples from six penicilliosis patients. There is no significant difference in detection performance between the previous study [14] and this modified method. This work determines the prevalence of active penicilliosis in Guangzhou and evaluates risk factors for penicilliosis in HIV-infected patients. Mp1p antigenaemia is usually found in patients with an active infection, which can be acute or reactivated. The prevalence of active and often disseminated disease is evaluated by the Mp1p antigenaemia test. Among HIV-infected patient serum samples collected during 2004–11, the average prevalence of active penicilliosis was 9.36% (761/8131). This result was different from Thuy Le’s report [7], in which prevalence was approximately 4.4% in AIDS patients in Vietnam. This discrepancy may be related to the different demographics among HIV populations and their exposure to P. marneffei. Another possible reason may be that our immunoassays are more sensitive than blood culture and other diagnostic methods. Mp1p antigenaemia was further confirmed by detection of GM with the Platelia Aspergillus kit. A good concordance rate between Mp1p and GM was observed, which is similar to that reported in Taiwan [15]. But we could not exclude other possible reasons for cross-reactivity in the GM assay, especially the use of piperacillin-tazobactam, amoxicillin-clavulanate [18,19], in HIV-infected patients with opportunistic infections. The prevalence of histoplasmosis, which causes a positive GM
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result [20], are relatively low in Guangdong, China [21]. Moreover, aspergillosis is rare in HIV-infected people [22]. As for cryptococcosis. which also may give rise to a positive GM result, we found that cryptococcosis patient sera were negative when tested by our Mp1p antigen assay. The trend of active penicilliosis over the 8 years in the study period was shown in Table 1. The prevalence increased to a peak in 2010 before decreasing in 2011. This behaviour may be related to HIV populations with varying disease stages. The increase of active penicilliosis was positively correlated with the proportion of HIV-infected patients at the AIDS stage, and negatively correlated with the proportion at the HIV infection stage. This result indicates that the prevalence of active penicilliosis is associated with CD4 count. HIV-infected patients with CD4 count <50 cells/μL are prone to developing coinfections with various opportunistic fungal infections. In this study, we found that the median CD4 count of patients with penicilliosis was 15 cells/μL (IQR 6–39 cells/μL), among whom 78.57% had a CD4 count of <50 cells/μL. Mp1p antigenaemia usually occurred when the CD4 count was <50 cells/μL. We stratified the CD4 count into five levels, and calculated the prevalence of penicilliosis in each level. Among those with a CD4 count of <50 cells/μL, 28.06% developed penicilliosis. In addition to the CD4 count, seasonality was reported to be associated with the prevalence of penicilliosis [7,9]. In Guangzhou, warm and humid weather is especially suitable for the growth of fungi. In this study, we studied the relationship among the Mp1p antigen, humidity and temperature. Our results show that the prevalence of active penicilliosis is associated with humidity, but not temperature. These results are in accord with those from a previous study [9]. Besides Mp1p antigen, Mp1p antibody should be detected for diagnosis of penicilliosis. We tried to determine this in all 8131 samples by double antigen sandwich ELISA [23], but obtained a low detection rate (data was not shown). Our considerations or desynchrony of antigen and antibody detection are as follows: (i) the different phases for appearance of antigen and antibody; (ii) an overwhelmingly high amount of antigen may consume most antibodies which influences antibody detection by immunological method; (iii) some patients may be not able to mount an adequate antibody response due to immunosuppression. Therefore, we would redesign the dynamics observation to characterize Mp1p antibody in penicilliosis. Our study has some limitations. First, few consecutive serum samples and CD4 count data were available, which prevented us from monitoring the temporal changes in Mp1p antigen, and their relationships with CD4 count. Second, specimens were collected from the outpatient clinics of GZCDC, where the clinical details including other opportunistic infections, antifungal treatments and prophylaxis might be incomplete. Third,
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in a retrospective study, reference standards such as those for culture, histology and cytology were hard to use to confirm Mp1p antigenaemia results. However, a diagnostic Platelia Aspergillus kit was used, and there was good accordance between the GM and Mp1p antigenaemia tests. Fourth, we did not differentiate penicilliosis from histoplasmosis, although there is very low prevalence of histoplasmosis in China [21]. In conclusion, the prevalence of penicilliosis was approximately 9.36% in Guangzhou, China during 2004–11. Most cases of penicilliosis occurred in patients with CD4 count of <50 cells/μL during the rainy season. As an important opportunistic infection in HIV-infected patients in Guangzhou, increased clinical vigilance for the diagnosis of penicilliosis is needed. Penicilliosis can be fatal without timely and appropriate treatment, especially in AIDS patients. Our immunoassay was high throughput and easy to perform, and would serve as a good serodiagnostic tool for penicilliosis in HIV-infected patients. Serum Mp1p antigen screening in HIV-infected patients, especially in those with CD4 count of <50 cells/μL, may prevent mortality associated with penicilliosis.
Transparency declaration The authors declare no conflicts of interest.
Acknowledgements The work is original and has been partially presented at the 23rd European Congress of Clinical Microbiology and Infectious Diseases (Berlin, Germany, 27–30 April 2013). This work was supported by grants from the National Natural Science Foundation of China (NSFC, 81101299), the Science and Technology Programme of Guangzhou, China (2012Y2-00021), the Project for Engineering Technology Research and Development Centre of Guangdong Province Universities and Colleges (GCZX-A1106), and the Health and Medical Research Fund, Hong Kong Special Administrative Region, China (13121342). We thank Professor K.-Y. Yuen for revising the manuscript. The authors also thank the staff and graduate students of Guangzhou Centre for Disease Control and Prevention and our laboratory for assistance with this study.
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Please cite this article in press as: Wang Y-F, et al., Serological surveillance for Penicillium marneffei infection in HIV-infected patients during 2004–2011 in Guangzhou, China, Clinical Microbiology and Infection (2015), http://dx.doi.org/10.1016/j.cmi.2014.12.014