Mariam Sarwar  ( Armed Forces Institute of Pathology (AFIP), National University of Medical Sciences, Rawalpindi, Pakistan )
Syed Adeel Hussain Gardezi  ( Armed Forces Institute of Pathology (AFIP), National University of Medical Sciences, Rawalpindi, Pakistan )
Gohar Zaman  ( Armed Forces Institute of Pathology (AFIP), National University of Medical Sciences, Rawalpindi, Pakistan )
Aamer Ikram  ( National Institute of Health (NIH), Islamabad, Pakistan )
Luqman Satti  ( Armed Forces Institute of Pathology (AFIP), National University of Medical Sciences, Rawalpindi, Pakistan )
Muhammad Tahir Khadim  ( Armed Forces Institute of Pathology (AFIP), National University of Medical Sciences, Rawalpindi, Pakistan )

Objective: To assess the utility of galactomannan and beta-D-glucan assays in the diagnosis of invasive aspergillosis in clinically suspected cases, and to compare their diagnostic potential to determine whether a combination of the two may result in an early and specific diagnosis.

Methods: The descriptive cross-sectional case-control study was conducted at the Armed Forces Institute of Pathology, Rawalpindi, Pakistan, from April 1, 2017, to March 31, 2018, and comprised serum samples from clinically suspected invasive aspergillosis patients and healthy controls. The sera were tested for galactomannan and beta-D-glucan detection. Proven, probable and possible categories of invasive aspergillosis according to European Organisation for Research and Treatment of Cancer / Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group criteria. Galactomannan antigen was detected using a one-stage immunoenzymatic sandwich microplate assay. Beta-D-Glucan antigen was detected using a protease zymogen-based colorimetric assay. Sensitivity and positive / negative likelihood ratio of both the cases and the controls were calculated and compared.

Results: Of the 178 subjects, 119(67%) were cases and 59(33%) were controls. Beta-D-glucan assay was more sensitive than galactomannan assay (91.6% versus 80.67%) whereas galactomannan assay was more specific than beta-D-glucan assay (86.44% versus 76.27%) in the diagnosis of invasive aspergillosis. The sensitivities of both assays decreased with decreasing probability of invasive aspergillosis, i.e., maximum sensitivities of both beta-D-glucan and galactomannan assays were for proven cases (100% versus 87.5%), followed by probable cases (89.29% versus 85.71%), and possible cases (91.57% versus 78.31%).

Conclusion: Both beta-D-glucan and galactomannan assays seemed to play an encouraging role in the diagnosis of invasive aspergillosis in high-risk clinically suspected cases, with the former assay being more sensitive and the latter assay being more specific.

Keywords: Invasive aspergillosis, Galactomannan, Beta-D-glucan. (JPMA 70: 442; 2020).

https://doi.org/10.5455/JPMA.1476

 

Introduction

 

Invasive Aspergillosis (IA) is an opportunistic mycosis with high mortality,¹ especially among patients of haematological malignancies, haematopoietic stem cell transplant (HSCT ) and solid organ transplant (SOT ) recipients.^2-4 Early diagnosis and prompt initiation of treatment are critical for IA management in these patients since it is a major cause of death in such populations.⁵ Delay in the reliable diagnosis is usually associated with fatal outcome as fu ngal proliferation becomes overwhelming or therapy may no longer remain successful.³ The invasive nature of the gold standard for diagnosis i.e. histopathology and culture are often precluded due to cytopenia or critical condition of the patients. This emphasises the need for non-invasive diagnostic procedures allowing easy and early diagnosis of IA. High mortality rates and limitations in diagnostic methods lead to a requirement for improved detection techniques for IA diagnosis⁶. Serological biomarkers like galactomannan (GM) and (1,3)-beta-D-glucan (BDG), therefore, can play a significant role for establishing a convenient diagnosis of this potentially fatal clinical condition. Over the past decades, patient population having IA risk factors has expanded significantly, and IA, since its associated with high morbidity and mortality, requires improved diagnostic modalities. GM is currently a commonly used method for IA diagnosis and has high specificity, whereas BDG has a high negative predictive value (NPV), making it quite useful to rule out IA rather than to confirm it.⁷ GM and BDG assays can also play an important role in IA diagnosis in non-neutropenic patients with underlying respiratory diseases without haematologic malignancy or previous SOT.⁸ GM, is a major aspergillus cell wall component released during the invasive disease. A double-sandwich enzymelinked immunosorbent GM assay has been approved and is widely used for IA surveillance in immunocompromised patients.² This diagnostic modality has a threshold for detection as low as 1.0 ng/mL of GM in serum compared to latex agglutination test with a threshold of 15ng/mL.^9,10 The mean time of detection of serum GM can often be 7-14 days before other diagnostic clues become apparent. It also offers monitoring of GM levels to allow the initiation of preemptive antifungal therapy before life-threatening infection occurs. Although initial studies assessed the performance characteristics of this assay with high sensitivity and specificity, more recent studies show significant performance variations.¹¹ BDG is the most important and abundant polysaccharide component of the cell wall of most fungi. While incorporated within the fungal cell wall, BDG typically exists as an insoluble structure. In the presence of blood or other body fluids, it transforms into single helix, triple helix (most commonly), or random coil forms and is rendered soluble.¹² The assays developed specifically to measure fungal beta-glucan typically use serum and rely on the activation of the Limulus Amoebocyte Lysate (LAL) clotting cascade as the principle of detection. The current study was planned to assess the effectiveness of GM and BDG detection in sera for IA diagnosis in clinically suspected cases. Both the assays were planned to be compared to study the diagnostic potential of both markers in various clinical cases and the utility of a combination of the two tests for an early and specific IA diagnosis.

 

Patients and Methods

 

The descriptive cross-sectional case-control study was conducted at the Armed Forces Institute of Pathology (AFIP), Rawalpindi, Pakistan, from April 1, 2017, to March 31, 2018, and comprised serum samples from clinically suspected IA patients and healthy controls. After permission from the institutional ethics review board, samples were collected from suspected IA patients (with or without radiological evidence) of either gender aged 4-82 years. Serum samples were collected after obtaining informed consent from all the patients and the healthy controls. The patient group had all three IA categories - proven, probable and possible - described by the European Organisation for Research and Treatment of Cancer / Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) criteria.¹³ Blood samples were collected in sterile clot-activator tubes. Serum was separated, divided into aliquots and stored at 2°C to 8°C for up to 48 hours until testing. GM antigen was detected using a one-stage immunoenzymatic sandwich microplate assay (Platelia Aspergillus: Bio-Rad, Marnes-la-Coquette, France), according to manufacturer's instructions. Positive, negative and cut-off controls were included in each assay. The serum samples and controls were treated with the addition of sample treatment solution, vortexed, heated for 3 minutes at 100OC using a water bath, and centrifuged at 10,000xg for 10 minutes. The supernatant was then tested with an enzyme immunoassay (EIA), reading the optical density of each well at 450nm (reference filter of 620/630nm). A result was considered positive with index value of ≥0.5, and <0.5 index value was considered negative. BDG antigen was detected using a protease zymogenbased colorimetric assay (Fungitell assay: Associates of Cape Cod), based upon a modification of the LAL pathway. According to manufacturer's instructions, serum samples were vortexed, treated with serum pretreatment reagent (0.25 molarity [M] potassium hydroxide [KOH] and 1.2 M potassium chloride [KCl]) and incubated for 10 min at 37OC. A kinetic colorimetric assay was performed and read at 405nm minus 490nm for 40 minutes at 37OC. The concentration of BDG in each sample was automatically calculated using a calibration curve prepared with standard solutions ranging from 6.25 to 100 pg/mL. The assays were performed in duplicate. BDG levels of >80 pg/mL were considered positive results, <60 pg/mL were considered negative, 6 0 - 7 9 pg /mL wer e c on s i de re d i n de termi n a te. Serological assays were performed among clinical cases with a spectrum of evidence of mycological infections. Statistical analysis was done, using SPSS 16.0 as the statistical tool, to determine sensitivity and positive likelihood ratio of all the three IA categories and specificity and negative likelihood ratio of the controls. Results were also evaluated to determine the significance of both serological assays done in combination.

 

Results

 

Of the 178 subjects, 119(67%) were cases and 59(33%) The median age of the patients was 33 years with an IQR of 32 years. Among the patients, 82(69%) were male and 37(31%) were female. Among the controls, 33(56%) were male and 26(44%) were female. The median age of controls was 30 years with an IQR of 35 years. The clinical history of the patients was noted (Table 1).

Among the patients, 8(6.7%) were in the proven category with histopathological evidence of IA, 28(23.5%) were in the probable category with growth on culture or fungal hyphae on direct microscopy, and 83(69.7%) were in the possible category with clinical features suggestive of IA with or without radiological features. Both GM and BDG assays were tested on patients in all the three categories as well as on the controls (Table 2).

BDG was more sensitive than GM assay (91.6% versus 80.67%) whereas GM was more specific than BDG (86.44% versus 76.27%). The sensitivities of both assays decreased with decreasing IA probability (Table 3).

Among the patients, 96(80.7%) were GM-positive, 23(19.3%) were GM-negative, while 109(91.6%) were BDG-positive, and 10(8.4%) were BDGnegative (Table 4).

 

Discussion

 

The presence of risk factors and clinical suspicion with other evidence of IA can be supported by either GM or BDG assay, but the diagnostic potential is improved if both assays are performed / interpreted simultaneously. Several studies have reported variable sensitivities and specificities of GM and BDG assays. One such study compared the two assays and found the GM assay to be more specific than BDG (97% versus 82%), and BDG more sensitive than GM (81% versus 49%), concluding that a combination of both tests could strengthen the diagnosis of IA.¹² Another study also reported higher sensitivity associated with BDG assay and higher specificity associated with GM assay for the diagnosis of IA.¹³ BDG assay is convenient to perform and is more sensitive, making it valuable as a screening tool. However, compared to GM, BDG bears higher cost and requires purchase of expensive glucan-free disposable materials. From a lab stand-point, BDG requires a batch of test requests, which poses limitations for the lab to entertain a single emergency request made by the clinicians.¹⁴ BDG assay can also yield false positive (FP) results, attributed to several factors other than invasive fungal infections (IFIs), including intravenous (IV ) use of antibiotics like amoxicillin-clavulanate and tazobactumpiperacillin,¹⁵ use of haemodialysis membranes in endstage renal disease (ESRD) patients,¹⁶ immunoglobulin (IGs) or albumin administration,¹⁷ blood cultures positive for bacteria,¹⁸ contamination of blood products by filtering through cellulose filters during manufacturing process,¹⁹ use of surgical gauzes containing glucan,²⁰ or mucositis etc.²¹ GM assay is easy to perform, gives early results, but it has a lesser sensitivity and higher specificity than the BDG assay FP results are also associated with the GM assay by release of GM antigen into circulation from factors other than IA, including foods, IV antibiotics such as tazobactum-piperacillin, dysfunction of gastrointestinal (GI) mucosal barrier releasing faecal GM into circulation,²² blood transfusion components etc.²³

 

Conclusion

 

GM was found to have high diagnostic specificity, while BDG displayed better sensitivity. Either test used alone carried cer tain level of diagnostic limitation. A combination of both assays would improve the diagnostic capacity.

 

Disclaimer: None.

Conflict of Interest: None.

Source of Funding: None.

 

References

 

1. Verdaguer V, Walsh TJ, Hope W, Cortez KJ. Galactomannan antigen detection in the diagnosis of invasive aspergillosis. Expert Rev Mol Diagn 2007;7:21-32.

2. Maertens J, Verhaegen J, Lagrou K, Van Eldere J, Boogaerts M. Screening for circulating galactomannan as a noninvasive diagnostic tool for invasive aspergillosis in prolonged neutropenic patients and stem cell transplantation recipients: a prospective validation. Blood 2001;97:1604-10.

3. Wright WF, Overman SB, Ribes JA. (1-3)-?-D-Glucan Assay: A Review of its Laboratory and Clinical Application. Lab Med 2011;42:679- 85.

4. Kawazu M, Kanda Y, Nannya Y, Aoki K, Kurokawa M, Chiba S, et al. Prospective comparison of the diagnostic potential of real-time PCR, double-sandwich enzyme-linked immunosorbent assay for galactomannan, and a (1-->3)-beta-D-glucan test in weekly screening for invasive aspergillosis in patients with hematological disorders. J Clin Microbiol 2004;42:2733-41.

5. Lamoth F, Calandra T. Early diagnosis of invasive mould infections and disease. J Antimicrob Chemother 2017;72(suppl 1):i19-i28.

6. Boch T, Spiess B, Cornely OA, Vehreschild JJ, Rath PM, Steinmann J, et al. Diagnosis of invasive fungal infections in haematological patients by combined use of galactomannan, 1,3-beta-D-glucan, Aspergillus PCR, multifungal DNA-microarray, and Aspergillus azole resistance PCRs in blood and bronchoalveolar lavage samples: results of a prospective multicentre study. Clin Microbiol Infect 2016;22:862-8.

7. Bassetti M, Peghin M, Vena A. Challenges and Solution of Invasive Aspergillosis in Non-neutropenic Patients: A Review. Infect Dis Ther 2018;7:17-27.

8. Prattes J, Flick H, Pruller F, Koidl C, Raggam RB, Palfner M, et al. Novel tests for diagnosis of invasive aspergillosis in patients with underlying respiratory diseases. Am J Respir Crit Care Med 2014;190:922-9.

9. Pfeiffer CD, Fine JP, Safdar N. Diagnosis of invasive aspergillosis using a galactomannan assay: a meta-analysis. Clin Infect Dis 2006;42:1417-27.

10. Pazos C, Ponton J, Del Palacio A. Contribution of (1->3)-beta Dglucan chromogenic assay to diagnosis and therapeutic monitoring of invasive aspergillosis in neutropenic adult patients: a comparison with serial screening for circulating galactomannan. J Clin Microbiol 2005;43:299-305.

11. Mennink-Kersten MA, Donnelly JP, Verweij PE. Detection of circulating galactomannan for the diagnosis and management of invasive aspergillosis. Lancet Infect Dis 2004;4:349-57.

12. Sulahian A, Porcher R, Bergeron A, Touratier S, Raffoux E, Menotti J, et al. Use and limits of (1-3)-beta-d-glucan assay (Fungitell), compared to galactomannan determination (Platelia Aspergillus), for diagnosis of invasive aspergillosis. J Clin Microbiol 2014;52:2328- 33.

13. Hachem RY, Kontoyiannis DP, Chemaly RF, Jiang Y, Reitzel R, Raad I. Utility of galactomannan enzyme immunoassay and (1,3) beta- D-glucan in diagnosis of invasive fungal infections: low sensitivity for Aspergillus fumigatus infection in hematologic malignancy patients. J Clin Microbiol 2009;47:129-33.

14. Fontana C, Gaziano R, Favaro M, Casalinuovo I, Pistoia E, Di Francesco P. (1-3)-?-D-Glucan vs Galactomannan Antigen in Diagnosing Invasive Fungal Infections (IFIs). Open Microbiol J 2012;6:70-3.

15. Marty FM, Lowry CM, Lempitski SJ, Kubiak DW, Finkelman MA, Baden LR. Reactivity of (1-->3)-beta-d-glucan assay with commonly used intravenous antimicrobials. Antimicrob Agents Chemother 2006;50:3450-3.

16. Kanda H, Kubo K, Hamasaki K, Kanda Y, Nakao A, Kitamura T, et al. Influence of various hemodialysis membranes on the plasma (1-- > 3 )- be t a-D - g l uc a n l ev e l. K i dn ey I nt 2 0 0 1 ; 60 : 31 9 - 2 3.

17. Ikemura K, Ikegami K, Shimazu T, Yoshioka T, Sugimoto T. Falsepositive result in Limulus test caused by Limulus amebocyte lysatereactive material in immunoglobulin products. J Clin Microbiol 1989;27:1965-8.

18. Pickering JW, Sant HW, Bowles CA, Roberts WL, Woods GL. Evaluation of a (1->3)-beta-D-glucan assay for diagnosis of invasive f un ga l i n fe c ti on s. J C l in Mi c rob i ol 2 00 5 ; 4 3 : 5 9 5 7 -6 2.

19. Nagasawa K, Yano T, Kitabayashi G, Morimoto H, Yamada Y, Ohata A, et al. Experimental proof of contamination of blood components by (1-->3)-beta-D-glucan caused by filtration with cellulose filters in the manufacturing process. J Artif Organs 2003;6:49-54.

20. Nakao A, Yasui M, Kawagoe T, Tamura H, Tanaka S, Takagi H. Falsepositive endotoxemia derives from gauze glucan after hepatectomy for hepatocellular carcinoma with cirrhosis. Hepatogastroenterology 1997;44:1413-8.

21. Ellis M, Al-Ramadi B, Finkelman M, Hedstrom U, Kristensen J, Ali- Zadeh H, et al. Assessment of the clinical utility of serial beta-Dglucan concentrations in patients with persistent neutropenic fever. J Med Microbiol 2008;57:287-95.

22. Ansorg R, van den Boom R, Rath PM. Detection of Aspergillus galactomannan antigen in foods and antibiotics. Mycoses 1997;40:353-7.

23. Martín-Rabadán P, Gijón P, Alonso Fernández R, Ballesteros M, Anguita J, Bouza E. False-positive Aspergillus antigenemia due to blood product conditioning fluids. Clin Infect Dis 2012;55:e22-7.