# Antemortem diagnostic tests for the detection of Aspergillus infection in birds: A systematic review

Sabrina Vieu, Jacques Guillot, François Beauveau

► To cite this version:

Sabrina Vieu, Jacques Guillot, François Beauveau. Antemortem diagnostic tests for the detection of Aspergillus infection in birds: A systematic review. Medical Mycology, 2024, 62 (11), 10.1093/mmy/myae112. hal-04828354

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# Antemortem diagnostic tests for the detection of *Aspergillus* infection in birds: a systematic review

3

4 **Short Title:** A systematic review of *aspergillus* infection in birds

5

6 **Authors and affiliations:**

7 Sabrina Vieu<sup>a,b,c*</sup> (DVM), Jacques Guillot<sup>a,c</sup> (DVM, PhD, Pr.), François Beaudou<sup>a,b</sup> (DVM,

8 PhD, Pr.)

9 <sup>a</sup> Oniris, VetAgroBio Nantes, 44300 Nantes, France

10 <sup>b</sup> Oniris, INRAE, BIOEPAR, 44300 Nantes, France

11 <sup>c</sup> IRF, IRCAT, Université d'Angers, 49000 Angers, France

12

13 * Correspondence: Sabrina Vieu +33 (0) 2 40 68 76 43 sabrina.vieu@yahoo.fr

14

15 **Key words:** diagnosis, aspergillosis, bird, sensitivity, specificity, systematic review.

16
## 17 ABSTRACT

Aspergillosis remains a common and life-threatening disease in captive and wild birds all over the world. Diagnosis is currently based on clinical signs or lesions, diagnostic imaging and a variety of biological tests. This systematic review aimed to compare the accuracy of antemortem diagnostic tests for *Aspergillus* infection in birds. According to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, a search was conducted on PubMed, Scopus, Web of Science, CAB until January 2024. The methodological quality was assessed with QUADAS 2 risk of bias tool. The thirteen studies, selected for the review, included results from a wide variety of birds (mainly Spheniciformes but also Falconiformes, Psittaciformes, and Galliformes) from wildlife rehabilitation centers, zoological parks or veterinary practices. *Aspergillus* infection was mainly confirmed by fungal culture and/or histopathology. Serum markers included *Aspergillus* components (galactomannan, β-D-glucan, mannoproteins and gliotoxin), anti-*Aspergillus* antibodies, 3-hydroxybutyrate as well as protein electrophoresis and acute phase molecules. Sensitivity and specificity displayed a large amount of variation despite threshold arrangement. Disparities in the number of individuals per study did not allow for reliable comparison. Platelia Ag Assay (Bio-Rad), the most commonly used test in the studies, demonstrated moderate specificity and low sensitivity. Overall, non-specific tests demonstrated more consistent performance, whereas specific tests showed greater variability. Based on current knowledge, none of these tests provide sufficient accuracy to reliably detect *Aspergillus* infection in birds in clinical practice.

## 39 LAY SUMMARY

Aspergillosis is a common and deadly bird disease. Diagnosing this disease is difficult before bird death. A review of 13 studies on various birds showed that current tests have inconsistent accuracy. No test reliably diagnoses aspergillosis in birds.
43 INTRODUCTION

44 *Aspergillus* fungi are common saprophytic molds which are responsible for opportunistic
45 infections referred as aspergillosis. Due to their ability to sporulate, their ubiquitous nature
46 and thermophily, *Aspergillus* fungi easily colonize the respiratory tract of birds and
47 aspergillosis is regularly reported in captive and wild birds all over the world.¹,² Severe
48 lesions, with acute and chronic forms are described in a wide range of avian species.³ In
49 poultry sector, aspergillosis is associated with mortality rates that fluctuate between 4.5% and
50 90%, and produces severe direct and indirect economic losses.⁴,⁵ Given the high mortality rate
51 of wild birds in captivity, aspergillosis is also a significant concern in zoological parks,
52 wildlife rehabilitation centers where it remains a major issue with reported mortality rates up
53 to 50% in captive Magellanic penguins (*Spheniscus magellanicus*), and captive birds of prey
54 used for falconry.⁶⁻¹⁰

55 Diagnosis of avian aspergillosis is currently based on clinical signs or lesions, diagnostic
56 imaging, and blood tests.²,¹¹⁻¹³ Direct visualization of lesions by endoscopy, associated with
57 histopathology and culture or postmortem is the only method of certifying the presence of
58 *Aspergillus* infection in animals.¹⁴ A thorough antemortem diagnosis is often difficult, owing
59 to the different forms of aspergillosis that may be also encountered: asymptomatic,
60 subclinical, acute or chronic forms.¹⁵ Consequently, expanding our understanding of avian
61 aspergillosis is essential not only for improving animal health and welfare, but also for
62 promoting the conservation of endangered bird species.¹,¹⁶,¹⁷ This progress is enabled by a
63 better knowledge of antemortem diagnostic tests for aspergillosis.

64 A large number of such tests are available, including both specific tests, which may be direct
65 or indirect for *Aspergillus* spp., and non-specific tests. Direct test and indirect tests are based
66 on the detection of antigens (e.g. galactomannan, b-D-glucan) and antibodies, respectively.¹⁸-
67 ²⁰ Non-specific tests include, among others, diagnostic imaging, biochemistry, and protein
68 electrophoresis.²¹⁻²³ However, none of these options is currently considered as a 'gold
69 standard' for reliable and effective diagnosis of aspergillosis in an antemortem context.
70 Literature data on comparative performance between and within studies are scarce. However,
71 having access to this information is essential for selecting the most suitable test for a
72 particular antemortem context.¹⁴

73 The objective of this work was to perform a systematic review to assess the accuracy of
74 available tests for antemortem detection of *Aspergillus* infection in birds.

75
## 76 Material and methods

### 77 Study design

This study was a systematic review of published studies since 1980. Findings have been reported according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 checklist.²⁴ Searches were performed in the following databases: Scopus, Web of Science, CAB (Commonwealth Agricultural Bureau) Abstracts and PubMed/MEDLINE. Search terms included: (Aspergillus OR aspergillosis) AND (birds OR bird OR avian OR aves OR poultry) AND (diagnostic OR diagnosis OR diagnose OR diagnoses OR test OR tests OR assay OR assays OR detection OR accuracy OR sensitivity OR specificity). The search strategy included articles published on the above-listed databases up to and including 08 January 2024.

### 88 Inclusion criteria

The criteria for study selection were set by three investigators, and took into account the key components of diagnostic test accuracy questions, which are the population (P), index test (I), and target condition (T).²⁵ On the basis of this type of questions, we searched databases using the following key criteria: The study population of interest included birds of any age (P) on which antemortem diagnosis tests were performed (I) clinically suspected, infected or inoculated by *Aspergillus* fungi (T). We only included articles written in English after 1980, published in peer-reviewed journals, and reporting sensitivity, specificity or information enabling their calculation.

### 98 Exclusion criteria

Articles were excluded if: (1) they were not written in English; (2) they were written before 01 January 1980 (3) relevant data could not be extracted; (4) they were conferences, reviews, case reports, letters to the editor or not peer reviewed (5) they did not report sensitivity, specificity or information enabling their calculation.

### 104 Data extraction and risk of bias

Data were extracted from the included studies by SV. All retrieved literature was imported into the Rayyan software (https://www.rayyan.ai/) for duplication removal and screening.
This tool enables blinded collaborative work and allows time saving for articles classification.
For each study the authors' name, title, journal and year of publication, abstract, were
documented on Rayyan software. The first screening step was based on the language, date,
type of article and species, filtering only relevant records and excluding irrelevant ones based
on the review question and eligibility criteria. One reviewer (SV) carried out the first step.
The remaining articles were all screened by the three reviewers (SV, JG and FB). During the
screening process, studies were included only if they adhered to the predefined inclusion
criteria. Any disagreements that occurred during this process were resolved via dialogue
between reviewers and discussed until consensus agreement was reached. The full texts of
chosen articles were scrutinized based on the previous established criteria, and they were
incorporated into the review if they satisfied all eligibility requirements. The collected
information were the year of study, number and species tested, reference standard and index
tests with respective thresholds if mentioned, index test sensitivity and specificity if available.
All information was collated in an Excel Spreadsheet (Excel®, Microsoft®Redmond, WA).
The risk of bias was conducted using the QUADAS-2 (Quality Assessment of Diagnostic
Accuracy Studies-2) developed for human diagnostic test accuracy.²⁶ This tool is based on a
form to fill in, which comprises four bias categories: (1) patient selection, (2) index test, (3)
reference standard, and (4) flow and timing. Each category underwent evaluation using a
specific question to assess the Risk of bias, and each criterion was rated as "high", "low" or
"unclear" of bias. Signaling questions were also included in the 4 categories to help judge the
risk of bias and were rated as “yes”, “no” or “unclear”. These questions highlight elements of
the study design that could introduce bias and are intended to assist reviewers in assessing the
risk of bias. Finally one question in each category was used to describe the concerns regarding
applicability and was rated as "high", "low" or "unclear".²⁶ The risk of bias was assessed by
two authors of this article (SV and FB). In case of non-agreement, a consensus was sought by
soliciting the third author (JG).

133

134

RESULTS

135

Study selection and characteristics

A summary of the data extraction is presented in **Figure 1**. A total number of 2865 articles
were imported into the Rayyan software, of which 922 were removed after deduplication. A
large portion of the studies resulting from the initial search was excluded through title,
136
137
138
139

abstract, language, year and type of publication, allowing to reduce from 1943 results to 62
articles. Four reports were not retrieved. Fifty-eight were read and screened for eligibility.

140

141

Specifically, two studies reported diseased and healthy birds. However, they did not report
enough precise data to calculate sensitivity and specificity.²⁷,²⁸ Thirteen studies were finally
included in the systematic review.

142

143

144

Main characteristics of the studies are summarized in **Table 1**. These studies were published between 2006 and 2023. Only two studies were experimental while the others were mainly case control designed. The samples originated from either biobanks or laboratories. Clinical records were directly obtained from veterinary clinics or wildlife rehabilitation centers to facilitate the categorization of the birds. Only biological tests studies answered criteria, in which serum, plasma or glottis mucus sample were analyzed. Analyses were sometimes performed on prolonged freeze samples.²⁹⁻³¹ Beside those providing sensitivity and specificity, additional six articles, retrieved from the research criteria, included sufficient data to retrospectively calculate the performance of the various tests.²⁰,²⁹⁻³³

145

146

147

148

149

150

151

152

153

154

Risk of bias

155

156

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

One signaling question (Did the study avoid inappropriate exclusions?) was removed from the tool because not enough details were given on each avian patient in the 13 articles to answer properly. The question (Are there concerns that the included patients do not match the review question?) belongs to concerns regarding applicability domain and was challenging to use in "Patient selection", because of the lack of information on each cases included. The risk of bias assessment for each study evaluated both its relevance to the research question and the methodology employed in the study.³⁴ Four studies were classified as having a high risk of bias for two categories (Figure 2).³¹,³³,³⁵,³⁶ One study was classified as having a high risk of bias for three categories, and thus may be considered "at risk of bias".³⁷ One study was classified with low risk of bias in all categories ²⁹ The combined risk of bias of all studies (Figure 3), illustrates that "Flow and timing" is often a category likely to be biased. The amount of time between sampling and testing of samples was sometimes imprecise, such as "stored for extended periods" which is a potential issue for test validation.²⁹ In several studies, a reference standard, such as histopathology or culture, could not be performed on all the animals as it was only performed on necropsied animals or following endoscopy procedure.²⁰,²⁹,³⁵,³⁸ No study received a "yes" for the signaling question "Did all patients receive the same reference standard?". For the other categories, ie. Patient selection, Index
172 test interpretation and Reference standard, the relative proportions of studies with a risk of bias rated as “low”, “unclear” and “high” were globally similar.

174

175 Studied population

176 A wide diversity of birds was obtained across the results, however
177 Spheniciformes<sup>20,31,33,35,36,39</sup>, Falconiformes<sup>32,37-39</sup> and Psittaciformes<sup>19,20,29,39</sup> were the most
178 represented with 6, 4, and 4 studies, respectively. According to the review, different types of
179 case were defined and retrieved such as proven case, possible, probable, or healthy/control
case. A large variability appeared between healthy/control groups, with a range of 6 to 146
180 birds,<sup>32,37</sup> and in diseased animals from 12 to 233 birds.<sup>20,38</sup> Variations in group size can
181 significantly impact test performance, and in small sample populations, sensitivity and
182 specificity may not be representative.

183 Most of the confirmed cases were certified by histopathology, fungal culture or necropsy findings. The criteria used to classify birds as healthy varied significantly across the selected studies. The existence of sub-clinically infected birds with *Aspergillus* spp. may impair the accuracy of the diagnostic. In two studies, healthy cases corresponded to birds with the absence of abnormalities at clinical examination and on biochemical or bloodwork analysis<sup>29,36</sup> or released birds after stay in rehabilitation center.<sup>36</sup> A detailed study described a discrimination based upon physical examination, endoscopy of the caudal thoracic air sacs, hematology, radiography, culture and cytology.<sup>29</sup>

192

193 Index tests

194 Index tests corresponded to a single test or a combination of tests (Table 2). Three studies
195 examined more than one index test.<sup>30,32,40</sup> Two studies evaluated at least one index test at
196 multiple thresholds.<sup>36,39</sup> Index tests included specific and non-specific tests. A first series of
197 specific tests allowed the direct detection of *Aspergillus* in birds by the dosage of antigens
198 (galactomannan, β-D-glucan or antigenic mannoproteins) or metabolites (gliotoxin).<sup>19,29-31,39</sup>
199 Other specific tests were indirect and based on the detection of antibodies against
200 *Aspergillus*.<sup>20,32,38</sup> Non-specific tests included physiological or global immunological
response to *Aspergillus* infection such as 3-hydroxybutyrate dosage and plasma protein
201 electrophoresis.<sup>30,33</sup> Inflammatory markers or innate immunity components (serum amyloid
202 A, ceruloplasmin, transferrin, haptoglobin, total plasma iron, and mannan-binding lectin)
203 were also used as non-specific tests.<sup>32,40</sup>
205 Test performance

206 The performance of 26 index tests was retrieved among the 13 selected studies (Table 2). All 207 95% confidence intervals for the tests performance were calculated from available data in 208 manuscripts. The sensitivity of all tests ranges from 7% to 97%, with a median of 70%, 209 whereas specificity ranges from 0% to 100%, with a median of 90%. The variation in 210 sensitivity and specificity was best viewed with individual studies, arranged by index test and 211 positivity threshold (Table 2). Sensitivity and specificity displayed a large amount of 212 variation despite threshold arrangement. Disparities in the number of individuals per study did 213 not allow for reliable comparison. Paired forest plots of sensitivity and specificity (Table 2) 214 revealed a high level of heterogeneity for results of the majority of tests. For instance, the 215 Platelia Ag Assay (Bio-Rad) was the most commonly used test in the studies, and 216 demonstrated specificity varying from 61 to 83 %, and sensitivity varying from 12% to 45 % 217 with a cut-off ≥ 1. Several studies did not show any false positive for different tests, resulting 218 in a 100% calculated specificity.31,32,40 Overall, non-specific tests demonstrated more 219 consistent performance, whereas specific tests showed greater variability. High values of 220 performance (ie. of sensitivity and specificity) did not mean that the corresponding studies 221 were per se at low risk of bias. As an illustration, the study from Arca-Ruibal et al. also 222 displayed a very high value of specificity (95%) but with a high risk of bias.37 By contrast, the 223 study from Reidy et al. was rated at a low risk of bias and also exhibited a specificity of 98% 224 in the detection of gliotoxin and bis(methyl)gliotoxin.29
225 DISCUSSION

226 The ability to make decisions on treatment and to detect changes in bird health status relies on
227 having accurate methods for the detection of the presence of *Aspergillus* infection. The
228 objective of the present systematic review was to assess the accuracy of available diagnostic
229 tests in birds. The main result of this review is that no recommendation can be provided for
230 the selection of a test when *Aspergillus* infection is suspected in a bird. Despite using a broad
231 search equation, only 13 articles with sufficient data on tests performance could be selected.
232 The limited number of available studies and the lack of standardized population samples
233 account for this situation.

234 Surprisingly, studies based on diagnostic imaging, a commonly used test to diagnose
235 aspergillosis, were excluded from the systematic review as these studies did not include
236 sensitivity and specificity.⁴¹⁻⁴⁴ Studies upon molecular detection using PCR assays (including
237 real-time PCR) were also lacking. PCR tests have been applied in experimentally infected
238 turkeys.¹⁸ This study did not include performance evaluation. PCR has also been used to
239 confirm the identification of *Aspergillus* isolates from captive birds.⁴⁵

240 As a part of the evidence-base medicine, assessing the risk of bias is crucial in evaluating
241 diagnostic test accuracy.²⁵ This systematic review, utilizing the QUADAS-2 tool, describes 13
242 studies with a moderate risk of bias. The QUADAS-2 tool is indeed commonly used in
243 systematic reviews to assess the risk of bias and relevance of primary diagnostic accuracy
244 studies in human medicine.³⁴ However, applying this tool to animal studies remains largely
245 subjective and challenging. The use of SYRCLE (SYstematic Review Centre for Laboratory
246 animal Experimentation) risk of bias tool for animal studies would have been interesting.⁴⁶
247 However, it can only be applied to assess experimental studies, of which there were only two
248 in the current review. While adhering to PRISMA guidelines for the objective assessment of
249 Risk of Bias, a notable level of subjectivity persists within this scoring framework. Studies
250 identified as having a high Risk of Bias should not be interpreted as low-quality evidence.

251 The latter is judged by other tools such as the GRADE system, which is difficult to apply to
252 diagnostic accuracy studies.⁴⁷ The inclusion of Risk of Bias assessment introduces additional
253 criteria for readers to utilize in guiding their interpretations.

254 Wide variations in test results were observed in the present review. This could be attributed to
255 the broad biological differences among avian species and inconsistent criteria used for the
256 definition of control or aspergillosis groups. A test performed in one species may not yield the
same results in another, making interpretation challenging and preventing the direct transposition of test results across species.

Reviewing individual study outcomes revealed variability in sensitivity or specificity estimates, underscoring the need to aggregate results through a meta-analysis to prevent over- or underestimating test performance based on a single study. However, the diverse range of outcome measures prevented the possibility of conducting a meta-analysis of the data.

In the present review, we were able to compare the performance of 26 index tests. The median across all tests was 70% and 90% for sensitivity and specificity, respectively. Wide variations were detected through the use of certain tests, with different risk of bias in the studies.

Among them, studies using galactomannan displayed a sensitivity varying from 7% to 86%.

This marker is usually measured for the diagnosis of invasive aspergillosis in human medicine, as it is a polysaccharide found in the cell wall of *Aspergillus* species released during the growth of the organism.<sup>48,49</sup> While the cut-off value in human medicine remains at 0.5, different index thresholds have been used in birds to define a positive galactomannan test, including 0.5, 0.7, 1.0, 1.5, and 2.0. The variability of the cut-off and of birds groups prevented a meaningful comparison between studies. Studies using β-D-glucan displayed a specificity of 93% and a sensitivity of 60%, with cut-off values of ≥461 pg/mL for seabirds and other birds, and ≥669 pg/mL for Japanese quails.<sup>19</sup> The β-D-glucan test is widely applied in human medicine to detect fungal infections. It measures a key polysaccharide released from fungal cell walls during growth.<sup>50</sup> Elevated levels are also observed in multiple fungal diseases, including invasive aspergillosis, *Pneumocystis jirovecii* pneumonia, *Pseudallescheria* infections, fusariosis, and candidiasis.<sup>51,52</sup> A study in broiler chickens with aspergillosis found no significant differences in β-D-glucan levels, while high readings (≥800 pg/mL) may stem from sample contamination, environmental exposure, or fungal colonization.<sup>53</sup>

One of the studies included in the review used the *Aspergillus* lateral-flow device (AspLFD, OLM diagnostics, Braintree, UK) for the detection of an early germ tube-specific extracellular mannoprotein. The study did not show any false negative results, resulting in a 100% calculated specificity in all comparisons, and a poor sensitivity of 47 to 76 %.<sup>31</sup> This result must be interpreted with caution because of the small number of samples and the use of the test in only one species (captive Gentoo penguins) in the study, which lacked a homogeneous reference standard and contributed to a significant level of bias. Another study, measuring gliotoxin and bis(methyl)gliotoxin levels in avian plasma, showed a median sensitivity of 70% and a specificity of 98%, with a low risk of bias in the study design.<sup>29</sup> However, the
detection of these metabolites requires expensive and inaccessible instrumentation, making it
difficult to use for routine analyses.

Indirect tests are based on the detection of antibodies. Cray et al. (2009) used an
unfractionated *Aspergillus* antigen (Immy®, Norman, OK, USA) with anti-chicken IgG to
create an ELISA.²⁰ The index of ≥1.4 was calculated based on the absorbance of the test
sample divided by the mean absorbance of negative control samples. This test was used in
two other studies from the same research group.³⁰,³² While using the same cut-off on different
cohorts, results remained not reliable with variable sensitivity (from 44% to 97%) and
specificity (from 21% to 100%). Another indirect test implied the use of Afmp1p, a
galactomannoprotein located in the cell wall of *A. fumigatus*.⁵⁴ An ELISA-based antibody
assay with Afmp1p was created and used in falcons. The test displayed a sensitivity of 75 %
and a specificity of 99%.³⁸ The study included only 12 individuals as true positives, but the
risk of bias remained low. The Anti-*Aspergillus* antibodies detection by double radial agar gel
immunodiffusion (Immy®, Norman, OK, USA) relies on the principle of immunodiffusion.⁵⁵
A sensitivity of 64% and a specificity of 95% were reported, but the study was associated
with a high risk of bias in patient selection.³⁵

Although specific tests did not allow a definitive antemortem diagnosis, various non-specific
markers have been investigated as alternatives. One of the study included in the review showed
that African penguins with aspergillosis had significantly elevated 3-hydroxybutyrate compared
with healthy birds, with average test performance (Se= 65%, Sp= 79%). This ketone body 3-β-
hydroxybutyrate, synthesized in the liver from fatty acids, serves as a crucial energy carrier to
peripheral tissues.⁵⁶ Since ketone bodies are synthesized during weight loss, additional analysis
integrating body score condition is essential to distinguish low scores that might cause false
positives. Combining 3-hydroxybutyrate with α2-globulin as an indirect test showed a
sensitivity of only 55% and a specificity of 98%.³⁰ This combination should be interpreted
carefully, as various factors can influence both parameters. Alpha2-globulin is found in protein
electrophoresis analysis which displayed various sensitivity (45-70%) and specificity (67-92%)
according to the protein analyzed (prealbumin, albumin, alpha2-globulin). Indeed, this test can
be affected by preanalytics and analytic variability such as electrophoresis system or prolonged
refrigeration, which was the case in some studies.²²,⁵⁷⁻⁵⁹ Ultimately, acute phase proteins were
used in two studies.³²,⁴⁰ While serum amyloid A displayed poor sensitivity (48.4%),³² it can
serve as a follow-up marker in birds due to its rapid changes in periodic samples, indicating a
positive response to treatment or the onset of sub-clinical disease.⁶⁰ One study reported serum
amyloid A values ranging from 2.3 to 137.5 mg/L (mean [SD], 47.7 +/- 29.7 mg/L) in 63 avian
patients with inflammatory disease, including falcons with chronic pododermatitis or fungal pneumonia and airsacculitis, who had persistently elevated serum amyloid A levels.⁶¹ Two studies included in this review reported contradictory data on haptoglobin concentrations in birds with aspergillosis, with cut-off values of ≥0.5 mg/mL and ≤0.044 mg/mL.³²,⁴⁰³,⁴⁰ These results were obtained in studies with a similar low risk of bias, and these discrepancies make the results of this test challenging to interpret. Concentration of haptoglobin may also increase in case of infectious bronchitis virus infection as part of the acute phase response in chicken.⁶²,⁶³

Other acute phase proteins, such as total iron-binding capacity (TIBC), unsaturated iron-binding capacity (UIBC), ceruloplasmin, and plasma iron levels, were also markedly increased. More particularly, the association of TIBC and ceruloplasmin reported good performance with 89% and 100% of sensitivity and specificity respectively. In the same study, mannan-binding lectin (MBL), a protein of the innate immune system displayed a sensitivity of 66% and a specificity of 74%, with a low risk of bias. However, given that the control group consisted in only 16 quails and that acute phase proteins can be elevated by other diseases, increased values of these markers should be interpreted with caution.⁴⁰

In birds, aspergillosis manifests in various forms that may necessitate different diagnostic methods depending on disease severity and extent. For example, a small granuloma may not be associated with the same markers as a large lesion in an air sac. In the 13 studies included in this review, the form of the disease was not always specified, thus test results may be affected. Proved disease requires histopathologic, cytopathologic, or direct microscopic confirmation of tissue invasion. The “gold standard” for diagnosing aspergillosis can be culture, but the development of a few colonies does not necessary confirm the infection due to the ubiquity of *Aspergillus* fungi, which is known to be contaminants. A diagnosis is more reliable when there is an abundant culture. However, a negative culture, especially from a less reliable sample like a tracheal swab, does not rule out aspergillosis.¹⁵ Histology can suggest aspergillosis, but due to the similarity of hyaline filamentous fungi in vivo, this method cannot identify the specific fungal species.⁶⁴,⁶⁵ A study revealed a 17% discordance between histologic analysis and fungal culture results upon fungi identification, highlighting the importance of obtaining both analyses.⁶⁶ Also, performing culture or histology requires an endoscopy. This procedure requires experience and can increase the risk of death due to an anesthesia in birds already weakened by aspergillosis. The limited accessibility of this procedure prevents veterinary practitioners from obtaining a rapid and efficient diagnosis without its use.
Despite the range of direct antemortem tests explored here that do not necessitate anesthesia in most of the case, none of them offer a conclusive diagnosis. Additionally, direct tests are largely inaccessible to veterinary practitioners. Only point-of-care direct tests could represent a promising solution to overcome this constraint.⁶⁷⁻⁶⁹ Most indirect tests are not commercially available, rendering them inaccessible and impractical for clinical use.³²,³⁸ The only available test lacks of precision and requires further investigation.³⁵ Indirect tests based on antibody detection may lead to poor performance when applied to birds with impaired immune response. Non-specific tests, even when used in combination, failed to be highly sensitive and specific, and then failed to provide a reliable diagnosis.

In conclusion, most of the studies included in this review were characterized by variable sample sizes, some inconsistencies in case qualification, and non-reproducible results due to the lack of standardized equipment availability. The performance of 26 index tests were compared. Among these tests, the detection of gliotoxin and bis(methyl)gliotoxin, along with the association of TIBC and ceruloplasmin were above the median performances, as reported in studies with a low risk of bias.

In such circumstances, additional prospective clinical trials with proper establishment of proven and control groups are required. Moreover the application of both reference standard and index test on all the population is strongly encouraged. The sample size must also be large enough to provide accurate estimations.

## Author contributions

Sabrina Vieu (Conceptualization, Writing original draft, Writing review & editing), Jacques Guillot (Supervision, Writing review), François Beaudeau (Conceptualization, Supervision, Writing review).

## Conflict of interest

The authors declare no conflicts of interest.

## REFERENCES
392 1. Seyedmousavi, S. et al. Aspergillus and aspergilloses in wild and domestic animals: a global health concern with parallels to human disease. *Med. Mycol.* **53**, 765–797 (2015).

394 2. Beernaert, L. A., Pasmans, F., Van Waeyenberghe, L., Haesebrouck, F. & Martel, A. Aspergillus infections in birds: a review. *Avian Pathol. J. WVPA* **39**, 325–331 (2010).

396 3. Arné, P., Risco-Castillo, V., Jouvion, G., Le Barzic, C. & Guillot, J. Aspergillosis in Wild Birds. *J. Fungi* **7**, 241 (2021).

398 4. Kalkayeva, D. et al. Epidemiological characteristics and financial losses due to avian aspergillosis in households in the Almaty region, Republic of Kazakhstan. *Front. Vet. Sci.* **10**, 1141456 (2023).

401 5. Arné, P. et al. Aspergillus fumigatus in Poultry. *Int. J. Microbiol.* **2011**, 746356 (2011).

402 6. Al Hemeiri, M. A., Mohteshamuddin, K., de la Torre, A. A., Degafa, B. A. & Ameni, G. A retrospective study on the health problems of falcons in al ain, United Arab Emirates. *Emir. J. Food Agric. EJFA* **34**, 51–58 (2022).

405 7. Melo, A. M. et al. Aspergillosis, Avian Species and the One Health Perspective: The Possible Importance of Birds in Azole Resistance. *Microorganisms* **8**, 2037 (2020).

407 8. Silva Filho, R. P. da et al. Incidence density, proportionate mortality, and risk factors of aspergillosis in magellanic penguins in a rehabilitation center from brazil. *J. Zoo Wildl. Med. Off. Publ. Am. Assoc. Zoo Vet.* **46**, 667–674 (2015).

410 9. Debergh, H. et al. Pulmonary Aspergillosis in Humboldt Penguins-Susceptibility Patterns and Molecular Epidemiology of Clinical and Environmental Aspergillus fumigatus Isolates from a Belgian Zoo, 2017-2022. *Antibiot. Basel Switz.* **12**, 584 (2023).

413 10. Yamada, K., Toyotome, T., Matsumoto, N. & Itoh, M. Autopsy imaging for aspergillosis in King Penguin, an economically valuable zoo animal. *J. Vet. Med. Sci.* **82**, 373–375 (2020).
416 11. Redig, P. 14 - Infectious Diseases, Fungal Diseases. in *Avian Medicine (Third Edition)*
417 (ed. Samour, J.) 434–521 (Mosby, 2016). doi:10.1016/B978-0-7234-3832-8.00014-6.

418 12. Elad, D. & Segal, E. Diagnostic Aspects of Veterinary and Human Aspergillosis. *Front. Microbiol.* **9**, 1303 (2018).
419

420 13. Desoubeaux, G., Cray, C. & Chesnay, A. Challenges to establish the diagnosis of
421 aspergillosis in non-laboratory animals: looking for alternatives in veterinary medicine
422 and demonstration of feasibility through two concrete examples in penguins and dolphins.
423 *Front. Cell. Infect. Microbiol.* **12**, (2022).

424 14. Savelieff, M. G., Pappalardo, L. & Azmanis, P. The current status of avian aspergillosis
425 diagnoses: Veterinary practice to novel research avenues. *Vet. Clin. Pathol.* **47**, 342–362
426 (2018).

427 15. Wellehan •, J. F. X. et al. CHAPTER 2 - Infectious disease. in *Current Therapy in Avian
428 Medicine and Surgery* (ed. Speer, B. L.) 22–106 (W.B. Saunders, 2016).
429 doi:10.1016/B978-1-4557-4671-2.00011-2.

430 16. Winter, D. J. et al. A single fungal strain was the unexpected cause of a mass aspergillosis
431 outbreak in the world's largest and only flightless parrot. *iScience* **25**, 105470 (2022).

432 17. Dornelles, G. et al. Harris' hawk (Parabuteo unicinctus) as a source of pathogenic human
433 yeasts: a potential risk to human health. *Future Microbiol.* **17**, 169–175 (2022).

434 18. Melloul, E. et al. Assessment of Aspergillus fumigatus burden in lungs of intratracheally-
435 challenged turkeys (*Meleagris gallopavo*) by quantitative PCR, galactomannan enzyme
436 immunoassay, and quantitative culture. *Comp. Immunol. Microbiol. Infect. Dis.* **37**, 271–
437 279 (2014).

438 19. Burco, J. D., Ziccardi, M. H., Clemons, K. V. & Tell, L. A. Evaluation of plasma (1-->3)
439 beta-D-glucan concentrations in birds naturally and experimentally infected with
440 aspergillus fumigatus. *Avian Dis.* **56**, 183–191 (2012).
20. Cray, C., Watson, T. & Arheart, K. L. Serosurvey and diagnostic application of antibody titers to Aspergillus in avian species. *Avian Dis.* **53**, 491–494 (2009).

21. Vorbrüggen, S., Bailey, T. & Krautwald-Junghanns, M.-E. Radiographic findings in raptors affected with a mycosis of the respiratory tract. *Tierarztl. Prax. Ausg. K Klientiere Heimtiere* **41**, 311–318 (2013).

22. Cray, C., Rodriguez, M. & Zaias, J. Protein electrophoresis of psittacine plasma. *Vet. Clin. Pathol.* **36**, 64–72 (2007).

23. Fischer, D. & Lierz, M. Diagnostic Procedures and Available Techniques for the Diagnosis of Aspergillosis in Birds. *J. Exot. Pet Med.* **24**, 283–295 (2015).

24. Page, M. J. *et al*. The PRISMA 2020 statement: An updated guideline for reporting systematic reviews. *Int. J. Surg. Lond. Engl.* **88**, 105906 (2021).

25. Sargeant, J. M. & O’Connor, A. M. Scoping Reviews, Systematic Reviews, and Meta-Analysis: Applications in Veterinary Medicine. *Front. Vet. Sci.* **7**, 11 (2020).

26. Whiting, P. F. *et al*. QUADAS-2: a revised tool for the quality assessment of diagnostic accuracy studies. *Ann. Intern. Med.* **155**, 529–536 (2011).

27. Desoubeaux, G. *et al*. Translational proteomic study to address host protein changes during aspergillosis. *PLoS ONE* **13**, e0200843 (2018).

28. German, A. C., Shankland, G. S., Edwards, J. & Flach, E. J. Development of an indirect ELISA for the detection of serum antibodies to Aspergillus fumigatus in captive penguins. *Vet. Rec.* **150**, 513–518 (2002).

29. Reidy, L. *et al*. Detection of gliotoxin but not bis(methyl)gliotoxin in plasma from birds with confirmed and probable aspergillosis. *J. Zoo Wildl. Med.* **53**, 60–69 (2022).

30. Desoubeaux, G., Rodriguez, M., Bronson, E., Sirpenski, G. & Cray, C. Application of 3-hydroxybutyrate measurement and plasma protein electrophoresis in the diagnosis of
465 aspergillosis in african penguins (*Spheniscus demersus*). *J. Zoo Wildl. Med. Off. Publ.*

466 *Am. Assoc. Zoo Vet.* **49**, 696–703 (2018).

467 31. Mota, S. M. *et al*. Application of a novel aspergillus lateral-flow device in the diagnosis

468 of aspergillosis in captive gentoo penguins (*Pygoscelis papua papua*). *J. Zoo Wildl. Med.*

469 *Off. Publ. Am. Assoc. Zoo Vet.* **54**, 360–366 (2023).

470 32. Fischer, D. *et al*. Comparison of diagnostic tools for the detection of aspergillosis in blood

471 samples of experimentally infected falcons. *Avian Dis.* **58**, 587–598 (2014).

472 33. Naylor, A. D., Girling, S. J., Brown, D., Crompton, C. G. & Pizzi, R. Plasma protein

473 electrophoresis as a prognostic indicator in Aspergillus species-infected Gentoo penguins

474 (*Pygoscelis papua papua*). *Vet. Clin. Pathol.* **46**, 605–614 (2017).

475 34. Zeng, X. *et al*. The methodological quality assessment tools for preclinical and clinical

476 studies, systematic review and meta-analysis, and clinical practice guideline: a systematic

477 review. *J. Evid.-Based Med.* **8**, 2–10 (2015).

478 35. Cabana, Â. L. *et al*. Serological monitoring of antibodies for an early diagnosis

479 of aspergillosis in captive penguins. *Pesqui. Veterinária Bras.* **35**, 573–578 (2015).

480 36. Cabana, A. L. *et al*. Applicability of the Platelia EIA® Aspergillus test for the diagnosis

481 of aspergillosis in penguins. *Braz. J. Biol. Rev. Brasleira Biol.* **79**, 169–173 (2019).

482 37. Arca-Ruibal, B. *et al*. Assessment of a commercial sandwich ELISA in the diagnosis of

483 aspergillosis in falcons. *Vet. Rec.* **158**, 442–444 (2006).

484 38. Wernery, U. *et al*. Serodiagnosis of aspergillosis in falcons (Falco spp.) by an Afmp1p-

485 based enzyme-linked immunosorbent assay. *Mycoses* **61**, 600–609 (2018).

486 39. Cray, C., Watson, T., Rodriguez, M. & Arheart, K. L. Application of galactomannan

487 analysis and protein electrophoresis in the diagnosis of aspergillosis in avian species. *J.*

488 *Zoo Wildl. Med. Off. Publ. Am. Assoc. Zoo Vet.* **40**, 64–70 (2009).
489 40. Goetting, V. et al. Inflammatory marker profiles in an avian experimental model of aspergillosis. *Med. Mycol.* **51**, 696–703 (2013).

491 41. Schwarz, T., Kelley, C., Pinkerton, M. E. & Hartup, B. K. Computed tomographic anatomy and characteristics of respiratory aspergillosis in juvenile whooping cranes. *Vet. Radiol. Ultrasound Off. J. Am. Coll. Vet. Radiol. Int. Vet. Radiol. Assoc.* **57**, 16–23 (2016).

495 42. Vorbrüggen, S., Bailey, T. & Krautwald-Junghanns, M.-E. Radiographic findings in raptors affected with a mycosis of the respiratory tract. *Tierarztl. Prax. Ausg. K Klientiere Heimtiere* **41**, 311–318 (2013).

498 43. Itoh, M. et al. Characteristic imaging findings of the respiratory system in penguins with suspected aspergillosis in an aquarium. *J. Vet. Med. Sci.* **82**, 1260–1266 (2020).

500 44. Rivas, A. E. et al. Standing computed tomography in nonanesthetized little penguins (*Eudyptula minor*) to assess respiratory system anatomy and monitor disease. *J. Zoo Wildl. Med. Off. Publ. Am. Assoc. Zoo Vet.* **50**, 396–404 (2019).

503 45. Talbot, J. J., Thompson, P., Vogelnest, L. & Barrs, V. R. Identification of pathogenic Aspergillus isolates from captive birds in Australia. *Med. Mycol.* **56**, 1038–1041 (2018).

505 46. Hooijmans, C. R. et al. SYRCLE's risk of bias tool for animal studies. *BMC Med. Res. Methodol.* **14**, 43 (2014).

507 47. Gopalakrishna, G. et al. Applying Grading of Recommendations Assessment, Development and Evaluation (GRADE) to diagnostic tests was challenging but doable. *J. Clin. Epidemiol.* **67**, 760–768 (2014).

510 48. Beauvais, A., Fontaine, T., Amanianda, V. & Latgé, J.-P. Aspergillus Cell Wall and Biofilm. *Mycopathologia* **178**, 371–377 (2014).
512 49. Latgé, J.-P., Beauvais, A. & Chamilos, G. The Cell Wall of the Human Fungal Pathogen Aspergillus fumigatus: Biosynthesis, Organization, Immune Response, and Virulence. *Annu. Rev. Microbiol.* **71**, 99–116 (2017).

515 50. Maertens, J., Theunissen, K., Lodewyck, T., Lagrou, K. & Van Eldere, J. Advances in the serological diagnosis of invasive Aspergillus infections in patients with haematological disorders. *Mycoses* **50 Suppl 1**, 2–17 (2007).

518 51. Onishi, A. et al. Diagnostic Accuracy of Serum 1,3-β-d-Glucan for Pneumocystis jiroveci Pneumonia, Invasive Candidiasis, and Invasive Aspergillosis: Systematic Review and Meta-Analysis. *J. Clin. Microbiol.* **50**, 7–15 (2012).

521 52. Cadena, J., Thompson, G. R. & Patterson, T. F. Aspergillosis: Epidemiology, Diagnosis, and Treatment. *Infect. Dis. Clin. North Am.* **35**, 415–434 (2021).

523 53. França, M., Cray, C. & Shivaprasad, H. L. Serologic testing for aspergillosis in commercial broiler chickens and turkeys. *Avian Dis.* **56**, 160–164 (2012).

525 54. Yuen, K.-Y. et al. Characterization of AFMP1: a Novel Target for Serodiagnosis of Aspergillosis. *J. Clin. Microbiol.* **39**, 3830–3837 (2001).

527 55. Immunodiffusion Tests for Aspergillosis. in *Serodiagnosis of Mycotic Diseases* (eds. Palmer, D., Kaufman, L., Kaplan, W. & Cavallaro, J.) 111–122 (1977).

529 56. Newman, J. C. & Verdin, E. β-Hydroxybutyrate: A Signaling Metabolite. *Annu. Rev. Nutr.* **37**, 51–76 (2017).

531 57. Cray, C., King, E., Rodriguez, M., Decker, L. S. & Arheart, K. L. Differences in protein fractions of avian plasma among three commercial electrophoresis systems. *J. Avian Med. Surg.* **25**, 102–110 (2011).

534 58. Roman, Y., Bomsel-Demontoy, M.-C., Levrier, J., Chaste-Duvernoy, D. & Jalme, M. S. Effect of hemolysis on plasma protein levels and plasma electrophoresis in birds. *J. Wildl. Dis.* **45**, 73–80 (2009).
537 59. Thorel, M., Roman, Y. & Leclerc, A. Plasma protein electrophoresis in the white stork (*Ciconia ciconia*): agreement between agarose gel versus capillary zone methods and development of reference intervals. *J. Zoo Wildl. Med. Off. Publ. Am. Assoc. Zoo Vet.* **55**, 330–340 (2024).

541 60. Rhim, H., Kim, M., Gim, S. & Han, J.-I. Diagnostic value of serum amyloid A in differentiating the inflammatory disorders in wild birds. *Front. Vet. Sci.* **11**, 1284113 (2024).

544 61. Caliendo, V., McKinney, P., Bailey, T., Kinne, J. & Wernery, U. Serum Amyloid A as an Indicator of Health Status in Falcons. *J. Avian Med. Surg.* **27**, 83–89 (2013).

546 62. Asasi, K., Mohammadi, A., Boroomand, Z., Hosseinian, S. A. & Nazifi, S. Changes of several acute phase factors in broiler chickens in response to infectious bronchitis virus infection. *Poult. Sci.* **92**, 1989–1996 (2013).

549 63. Nazifi, S., Tabande, M. R., Hosseinian, S. A., Ansari-Lari, M. & Safari, H. Evaluation of sialic acid and acute-phase proteins (haptoglobin and serum amyloids A) in healthy and avian infection bronchitis virus-infected chicks. *Comp. Clin. Pathol.* **20**, 69–73 (2011).

552 64. Rickerts, V. et al. Comparison of Histopathological Analysis, Culture, and Polymerase Chain Reaction Assays to Detect Invasive Mold Infections from Biopsy Specimens. *Clin. Infect. Dis.* **44**, 1078–1083 (2007).

555 65. Kung, V. L., Chernock, R. D. & Burnham, C.-A. D. Diagnostic accuracy of fungal identification in histopathology and cytopathology specimens. *Eur. J. Clin. Microbiol. Infect. Dis.* **37**, 157–165 (2018).

558 66. Lee, S. et al. Discrepancy between histology and culture in filamentous fungal infections. *Med. Mycol.* **48**, 886–888 (2010).

560 67. Aerts, R. et al. Point-of-care testing for viral-associated pulmonary aspergillosis. *Expert Rev. Mol. Diagn.* **24**, 231–243 (2024).
68. Jenks, J. D., Miceli, M. H., Prattes, J., Mercier, T. & Hoenigl, M. The Aspergillus Lateral Flow Assay for the Diagnosis of Invasive Aspergillosis: an Update. *Curr. Fungal Infect. Rep.* **14**, 378–383 (2020).

69. Calero, A. L. et al. Comparison of the Performance of Two Galactomannan Detection Tests: Platelia Aspergillus Ag and Aspergillus Galactomannan Ag Virclia Monotest. *Microbiol. Spectr.* **10**, e0262621 (2022).

569 **FIGURE LEGEND**

570 **Figure 1:** PRISMA flow chart of the systematic review method applied to the diagnostic accuracy tests for *Aspergillus* spp. infection in birds.

573 **Figure 2:** Illustration of individual study risk of bias from the QUADAS-2 form for the studies included in the systematic review. A study is listed only once even if it included multiple tests. "High" means high risk of bias, "Unclear" means unclear risks of bias and "Low" means a low risk of bias. Plot from by robvis tool.

578 **Figure 3:** Combined risk of bias assessment from the QUADAS-2 form to illustrate the overall study risk of bias of the systematic review method applied to the diagnostic accuracy test in birds. Plot from by QUADAS-2 excel template.
<table><thead><tr><td>Ref</td><td>Birds origin</td><td>n (species, order or family)</td><td>n control group (samples precision if repeated measures)</td><td>n confirmed group (samples)</td><td>n probable group (samples)</td><td>n possible group (samples)</td><td>Biological sample</td><td>Confirmation of aspergillosis cases</td></tr></thead><tbody><tr><td>Arca-Ruibal B. et al. 2006</td><td>Cases from veterinary practice</td><td>192 Falconiformes</td><td>142 (182)</td><td>50 (90)</td><td>0</td><td>0</td><td>S</td><td>Fungal culture or histopathology</td></tr><tr><td>Cray C., et al. 2009</td><td>Laboratory received sample</td><td>1314 Psittaciformes, Falconiformes, Strigiformes, Sphenisciformes, Anseriformes, Galliformes, Columbiformes, Galliformes, Charadiformes, Passeriformes, Caprimulgiformes, Coraciiformes, Musophagiformes, Pelecaniformes, Ciconiiformes</td><td>70</td><td>57</td><td>176 (defined by "cumulative data and observations strongly consistent with aspergillosis")</td><td>0</td><td>P</td><td>Histopathology</td></tr><tr><td>Cray C., et al. (Rodriguez) 2009</td><td>Laboratory received sample</td><td>128 Falconiformes, Psittaciformes, Anseriformes, Galliformes, Spheniciformes</td><td>72</td><td>56</td><td>0</td><td>0</td><td>P</td><td>Histopathology</td></tr><tr><td>Burco J. D., et al. 2012</td><td>Cases from veterinary hospital, wildlife center and experiment (quail raised and hatch at university)</td><td>178<sup>2</sup> "pet" (Falconiformes, Strigiformes, Accipitriformes, Psittaciformes, Ramphastidae), "Seabird" (Gaviiformes, Podicipediformes, Alcidae, Phalacrocoracidae), Galliformes including chicken (Gallus Gallus) and japanese quail (Coturnix japonica)</td><td>9 SPF chicken + 54 non-SPF chicken + 23 control quail + 29 negative seabirds (38 samples)</td><td>15 seabirds (22 samples) + 19 pet + 17 quails</td><td>0</td><td>0</td><td>P</td><td>Histopathology and culture or necropsy lesions</td></tr><tr><td>Goetting V., et al. 2013</td><td>Experimental protocole</td><td>86 Japanese quail (Coturnix japonica)</td><td>16</td><td>70</td><td>0</td><td>0</td><td>P</td><td>Fungal culture on all birds and 28 with histopathology</td></tr><tr><td>Fischer D., et al. 2014</td><td>Experimental protocole</td><td>36 gyr-saker hybrid falcons (Falco rusticolus x Falco cherrug)</td><td>6</td><td>30</td><td>0</td><td>0</td><td>S or P</td><td>Post-mortem examination, only 14 falcons with observed aspergillosis granulomas confirmed</td></tr></tbody></table>
<table><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>by fungal culture and histopathology</td></tr><tr><td>Cabana Â. L., et al. 2015</td><td>Cases from Rehabitlitation center</td><td>134 Magellanic penguins (Spheniscus magellanicus)</td><td>101</td><td>33</td><td>0</td><td>0</td><td>S</td><td>Post-mortem examinations with Fungal culture and histopathological examination</td></tr><tr><td>Naylor A. D., et al. 2017</td><td>Banked sample from RZSS Edinburgh Zoo</td><td>183² Gentoo penguins (Pygoscelis papua papua)</td><td>82 (146)</td><td>34 (37)</td><td>0</td><td>0</td><td>P</td><td>Postmortem examination with histology and Fungal culture</td></tr><tr><td>Wernery U., et al. 2018</td><td>Experimental protocole and samples from veterinary clinic</td><td>141 Falconiformes</td><td>129</td><td>12</td><td>0</td><td>0</td><td>S</td><td>Postmortem examination with histology and Fungal culture</td></tr><tr><td>Desoubeaux G., et al. 2018</td><td>Laboratory received sample</td><td>172 African penguins (Spheniscus demersus)</td><td>29 inflammatory control and 96 clinically normal birds</td><td>7</td><td>15 (defined by positive fungal cultures in conjunction with clinical signalment and corroborative imaging investigations)</td><td>25 (defined by clinical signs of aspergillosis, no positive fungal cultures or histopathology evidences, no available imaging findings, and positive response to antifungal treatment)</td><td>P</td><td>Histopathology</td></tr><tr><td>Cabana A. L., et al. 2019</td><td>Cases from Rehabitlitation center</td><td>52 Magellanic penguins (Spheniscus magellanicus)</td><td>23</td><td>29</td><td>0</td><td>0</td><td>S</td><td>Fungal culture and histopathology</td></tr></table>
<table><tr><td>Reidy L., et al. 2022</td><td>banked sample AZA SSP Program</td><td>98 Spheniciformes, Psittaciformes, Falconiformes, Cathartiformes, Galliformes, Columbiformes, Gruiformes, Bucerotiformes</td><td>51</td><td>23</td><td>24 (defined by aspergillosis clinical signs, radiography sign of aspergillosis and positive response to antifungal treatment)</td><td>0</td><td>P</td><td>Histopathology</td></tr><tr><td>Mota S. M., et al. 2023</td><td>banked sample and prospective study at RZSS Eindburgh Zoo</td><td>38 Gentoo penguins (Pygoscelis papua papua)</td><td>24 (24 plasma and 14 swabs)</td><td>7 (5 plasma and 2 swabs)</td><td>4 (4 plasma and 4 swabs, defined by positive fungal culture, corroborative imaging investigations, or both)</td><td>6 (6 plasma and 6 swabs, defined by positive response to antifungal treatment, no positive fungal cultures, histopathology, or imaging findings available)</td><td>P and glottis mucus</td><td>Aspergillosis clinical signs and antifungal treatment response</td></tr></table>

**Table 1:** Study characteristics of the included studies in the systematic review of antemortem test for the diagnosis of clinically suspected, infected or inoculated birds by *Aspergillus spp.* Characteristics include primary author and year of publication, cases origin, bird order, type and groups of comparison, sample type, reference standard.

<footnote n="2">² birds with repeated samples; S: Serum; P: Plasma; RZSS: Royal Zoological Society of Scotland; AZA SSP: Associations of Zoos and Aquarium Species Survival Plan; SPF: Specific-Pathogen-Free</footnote>
<table><thead><tr><th></th><th>Index test</th><th>Reference</th><th>TP</th><th>FN</th><th>FP</th><th>TN</th><th>Groups</th><th>Cut-off</th><th>Unit</th><th>Sensitivity (95%CI)</th><th>Specificity (95% CI)</th></tr></thead><tbody><tr><td rowspan="14">Specific tests</td><td rowspan="14">Direct tests</td><td>β-D-glucan assay (Fungitell)</td><td>Burco J. D., et al. 2012</td><td>58</td><td></td><td>142</td><td></td><td></td><td>> 461</td><td>pg/dL</td><td>0.60 (0.47,0.73)</td><td>0.93 (0.88,0.97)</td></tr><tr><td rowspan="10">Galactomannan (Platelia ELISA kit)</td><td>Cabana A. L., et al. 2019</td><td>29</td><td></td><td>23</td><td></td><td></td><td>>0.5</td><td>Index</td><td>0.86 (0.74,0.99)</td><td>0.26 (0.08,0.44)</td></tr><tr><td>Cray C., et al. (Rodriguez) 2009</td><td>38</td><td>18</td><td>19</td><td>53</td><td></td><td>>0.5</td><td>Index</td><td>0.67 (0.55,0.79)</td><td>0.73 (0.63,0.83)</td></tr><tr><td>Fischer D., et al. 2014</td><td>2</td><td>28</td><td>0</td><td>6</td><td>F</td><td>>0.7</td><td>Index</td><td>0.07 (0.00,0.16)</td><td>1.00 (1.00,1.00)</td></tr><tr><td>Desoubeaux G., et al. 2018</td><td>11</td><td>36</td><td>96</td><td>0</td><td></td><td>>0.7</td><td>Index</td><td>0.23 (0.11,0.36)</td><td>0.00 (0.00,0.00)</td></tr><tr><td>Arca-Ruibal B. et al. 2006</td><td>6</td><td>44</td><td>7</td><td>13</td><td>5</td><td>>1.0</td><td>Index</td><td>0.12 (0.03,0.21)</td><td>0.95 (0.92,0.99)</td></tr><tr><td>Cabana A. L., et al. 2019</td><td>29</td><td></td><td>23</td><td></td><td></td><td>>1.0</td><td>Index</td><td>0.45 (0.27,0.63)</td><td>0.61 (0.41,0.81)</td></tr><tr><td>Cray C., et al. (Rodriguez) 2009</td><td>56</td><td></td><td>72</td><td></td><td></td><td>>1.0</td><td>Index</td><td>0.39 (0.26,0.52)</td><td>0.83 (0.74,0.92)</td></tr><tr><td>Cabana A. L., et al. 2019</td><td>29</td><td></td><td>23</td><td></td><td></td><td>>1.5</td><td>Index</td><td>0.41 (0.23,0.59)</td><td>0.83 (0.67,0.98)</td></tr><tr><td>Cabana A. L., et al. 2019</td><td>29</td><td></td><td>23</td><td></td><td></td><td>>2.0</td><td>Index</td><td>0.35 (0.17,0.52)</td><td>0.91 (0.80,1.03)</td></tr><tr><td rowspan="3">Aspergillus lateral-flow device (AspLFD)</td><td>Mota S. M., et al. 2023</td><td>4</td><td>8</td><td>0</td><td>14</td><td>C</td><td></td><td></td><td>0.47 (0.23,0.71)</td><td>1.00 (1.00,1.00)</td></tr><tr><td>Mota S. M., et al. 2023</td><td>6</td><td>6</td><td>0</td><td>14</td><td>D</td><td></td><td>Visualization of precipitation line</td><td>0.50 (0.22,0.78)</td><td>1.00 (1.00,1.00)</td></tr><tr><td>Mota S. M., et al. 2023</td><td>9</td><td>3</td><td>0</td><td>14</td><td>E</td><td></td><td></td><td>0.76 (0.56,0.97)</td><td>1.00 (1.00,1.00)</td></tr><tr><td rowspan="2">Gliotoxin and Bis(methyl)gliotoxin detection by LC-MS/MS</td><td>Reidy L., et al. 2022</td><td>16</td><td>7</td><td>1</td><td>50</td><td>A</td><td></td><td>3</td><td>ng/ml</td><td>0.70 (0.51,0.88)</td><td>0.98 (0.94,1.02)</td></tr><tr><td>Reidy L., et al. 2022</td><td>27</td><td>20</td><td>1</td><td>50</td><td>B</td><td></td><td>3</td><td>ng/ml</td><td>0.57 (0.43,0.71)</td><td>0.98 (0.94,1.01)</td></tr><tr><td>Aspergillus fumigatus (ID) IMMY®</td><td>Cabana Å. L., et al. 2015</td><td>21</td><td>12</td><td>5</td><td>96</td><td></td><td></td><td></td><td>Visualization of precipitation line</td><td>0.64 (0.47,0.80)</td><td>0.95 (0.91,0.99)</td></tr><tr><td rowspan="10">Indirect tests</td><td rowspan="5">Afmp1p-based ELISA</td><td>Wernery U., et al. 2018</td><td>12</td><td></td><td>129</td><td></td><td></td><td>0.407</td><td>Absorbance (nm)</td><td>0.75 (0.51,1.00)</td><td>0.99 (0.98,1.01)</td></tr><tr><td>Fischer D., et al. 2014</td><td>2</td><td>28</td><td>0</td><td>6</td><td>F</td><td>>1.4</td><td>Index</td><td>0.97 (0.90,1.00)</td><td>0.33 (0.00,0.71)</td></tr><tr><td>Desoubeaux G., et al. 2018</td><td>43</td><td>4</td><td>76</td><td>20</td><td></td><td>>1.4</td><td>Index</td><td>0.91 (0.84,0.99)</td><td>0.21 (0.13,0.29)</td></tr><tr><td>Cray C., et al. 2009</td><td>25</td><td>32</td><td>22</td><td>48</td><td>A</td><td>>1.4</td><td>Index</td><td>0.44 (0.31,0.57)</td><td>0.69 (0.58,0.79)</td></tr><tr><td>Cray C., et al. 2009</td><td>127</td><td>106</td><td>22</td><td>48</td><td>B</td><td>>1.4</td><td>Index</td><td>0.55 (0.48,0.61)</td><td>0.69 (0.58,0.79)</td></tr><tr><td rowspan="10">Aspergillus antibody ELISA</td><td>Galactomannan and protein electrophoresis</td><td>Cray C., et al. (Rodriguez) 2009</td><td>56</td><td></td><td>72</td><td></td><td></td><td>GM >0.5 and increase in any globulin fraction > 20%</td><td></td><td>0.89 (0.81,0.97)</td><td>0.48 (0.36,0.60)</td></tr><tr><td>Prealbumin + Albumin</td><td>Naylor A. D., et al. 2017</td><td></td><td></td><td></td><td></td><td></td><td>>26.19</td><td>(g/L)</td><td>0.70 (0.53,0.84)</td><td>0.77 (0.69,0.83)</td></tr><tr><td>Albumin-to-Globulin Ratio</td><td>Naylor A. D., et al. 2017</td><td>37</td><td></td><td>146</td><td></td><td></td><td>>0.96</td><td>ratio</td><td>0.70 (0.53,0.84)</td><td>0.75 (0.67,0.82)</td></tr><tr><td>Albumin</td><td>Naylor A. D., et al. 2017</td><td></td><td></td><td></td><td></td><td></td><td>>20.47</td><td>(g/L)</td><td>0.70 (0.53,0.84)</td><td>0.73 (0.65,0.80)</td></tr><tr><td>Albumin</td><td>Naylor A. D., et al. 2017</td><td></td><td></td><td></td><td></td><td></td><td>>37.34</td><td>% of total proteins</td><td>0.70 (0.53,0.84)</td><td>0.67 (0.59,0.75)</td></tr><tr><td>EPH: any globulin fraction</td><td>Cray C., et al. (Rodriguez) 2009</td><td>56</td><td></td><td>72</td><td></td><td></td><td>>20% compared to species reference values</td><td></td><td>0.73 (0.61,0.85)</td><td>0.70 (0.59,0.81)</td></tr><tr><td>α2-Globulin</td><td>Desoubeaux G., et al. 2018</td><td></td><td></td><td></td><td></td><td></td><td>> 1.16</td><td>g/dL</td><td>0.45 (0.34,0.58)</td><td>0.92 (0.86,0.95)</td></tr><tr><td>α2-Globulin and 3-hydroxybutyrate</td><td>Desoubeaux G., et al. 2018</td><td>47</td><td></td><td>96</td><td></td><td></td><td>> 1.16 g/dL and 0.94 mM/L</td><td></td><td>0.55 (0.51,0.59)</td><td>0.98 (0.94,0.99)</td></tr><tr><td>3-Hydroxybutyrate</td><td>Desoubeaux G., et al. 2018</td><td></td><td></td><td></td><td></td><td></td><td>> 0.94</td><td>mM/L</td><td>0.65 (0.50,0.76)</td><td>0.79 (0.62,0.88)</td></tr><tr><td rowspan="2">Serum amyloid A</td><td>Fischer D., et al. 2014</td><td>18</td><td>9</td><td>0</td><td>6</td><td>F</td><td>>48.4**</td><td>mg/mL</td><td>0.56 (0.37,0.74)</td><td>1.00 (1.00,1.00)</td></tr><tr><td>Goetting V., et al. 2013</td><td>86</td><td></td><td>16</td><td></td><td></td><td>>0.044</td><td>mg/mL</td><td>0.75 (0.65,0.84)</td><td>0.56 (0.45,0.66)</td></tr><tr><td>Haptoglobin</td><td>Fischer D., et al. 2014</td><td>19</td><td>8</td><td>0</td><td>6</td><td>F</td><td>>0.5**</td><td>mg/mL</td><td>0.70 (0.53,0.88)</td><td>1.00 (1.00,1.00)</td></tr><tr><td>Unsaturated iron-binding capacity (UIBC)</td><td>Goetting V., et al. 2013</td><td></td><td></td><td></td><td></td><td></td><td>> 13.5</td><td>µg/dL</td><td>0.80 (0.71,0.88)</td><td>0.90 (0.83,0.96)</td></tr><tr><td>Plasma iron</td><td>Goetting V., et al. 2013</td><td></td><td></td><td></td><td></td><td></td><td>> 16.4</td><td>µg/dL</td><td>0.71 (0.62,0.81)</td><td>0.63 (0.53,0.73)</td></tr><tr><td>Total iron-binding capacity (TIBC)</td><td>Goetting V., et al. 2013</td><td></td><td></td><td></td><td></td><td></td><td>> 39.0</td><td>µg/dL</td><td>0.76 (0.67,0.85)</td><td>1.00 (1.00,1.00)</td></tr><tr><td>Ceruloplasmin (Cer)</td><td>Goetting V., et al. 2013</td><td>86</td><td></td><td>16</td><td></td><td></td><td>> 28.0</td><td>umole ODA/min.*L</td><td>0.72 (0.62,0.81)</td><td>1.00 (1.00,1.00)</td></tr><tr><td>Mannan-Binding Lectin/complement</td><td>Goetting V., et al. 2013</td><td></td><td></td><td></td><td></td><td></td><td>> 1.75</td><td>serum dilution</td><td>0.66 (0.56,0.76)</td><td>0.74 (0.64,0.83)</td></tr><tr><td>TIBC + Ceruloplasmin</td><td>Goetting V., et al. 2013</td><td></td><td></td><td></td><td></td><td></td><td>TIBC > 39.0 Or Cer > 28.0</td><td></td><td>0.89 (0.82,0.96)</td><td>1.00 (1.00,1.00)</td></tr><tr><td>TIBC + UIBC</td><td>Goetting V., et al. 2013</td><td></td><td></td><td></td><td></td><td></td><td>TIBC > 39.0 Or UIBC > 13.5</td><td></td><td>0.89 (0.82,0.96)</td><td>0.90 (0.83,0.96)</td></tr><tr><td>UIBC + Ceruloplasmin</td><td>Goetting V., et al. 2013</td><td></td><td></td><td></td><td></td><td></td><td>UIBC > 13.5 Or Cer > 28.0</td><td></td><td>0.86 (0.78,0.93)</td><td>0.90 (0.83,0.96)</td></tr></tbody></table>

A: Control VS Confirmed; B: Control VS Confirmed + Probable; C: Control VS all group plasma, D: Control VS all group swab; E: Control VS all group plasma or swab; F= Adult + Juvenile control VS Adult + Juvenile confirmed

EPH: Heparinized electrophoresis; ODA = oxidation of o-dianisidine

<figure><img src="image_2.png" /></figure>
**Table 2:** Forest plots of sensitivity and specificity of the antemortem index tests for the diagnosis of clinically suspected, infected or inoculated birds by *Aspergillus* spp.. Each study is named with the first author's last name and the year of publication. The cut-off and test positive threshold are included. A square indicates each individual study sensitivity and specificity with 95% confidence intervals depicted by the horizontal line. All confidence intervals were calculated. Sensitivity and Specificity in gray were not initially provided and calculate with available data in studies. The number of samples falling in the true positive (TP), false positive (FP), true negative (TN) and false negative (FN) categories are listed.
<figure><img src="image_3.png" /><figcaption>Figure 1</figcaption></figure>

140x198mm (300 x 300 DPI)
<figure><img src="image_4.png" /><figcaption>Figure 2
350x389mm (300 x 300 DPI)</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Figure 3
121x43mm (300 x 300 DPI)</figcaption></figure>