Filtration-based culture methods improve recovery of fungal pathogens in respiratory specimens

Diagnostic Microbiology and Infectious Disease 56 (2006) 221 – 223 www.elsevier.com/locate/diagmicrobio

Filtration-based culture methods improve recovery of fungal pathogens in respiratory specimensB,BB Chadi M. El Saleeby a,d,e, Ginger R. Jamisonb, Stanley B. Poundsc, Carolyn B. Hewitt b, Randall T. Haydenb,d,4 a

Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, TN 38105-2794, USA b Department of Pathology, St. Jude Children’s Research Hospital, Memphis, TN 38105-2794, USA c Department of Biostatistics, St. Jude Children’s Research Hospital, Memphis, TN 38105-2794, USA d Department of Pediatrics, University of Tennessee Health Science Center, Memphis, TN, USA e Department of Pediatrics, Harvard Medical School, Massachusetts General Hospital for Children, Massachusetts General Hospital, Boston, MA 02114, USA Received 17 November 2005; accepted 13 April 2006

Abstract We describe a filtration-based culture method to enhance sensitivity of routine culture for recovery of fungi from respiratory specimens. The new method resulted in an 8.3% ( P = 0.0039) increase in recovery for all organisms and a 6.4% ( P = 0.0391) for Candida species when compared to the conventional culture technique. D 2006 Elsevier Inc. All rights reserved. Keywords: Mycosis; Filtration; Culture; Respiratory infections

The respiratory tract is a common site of invasive fungal infections (Chen et al., 2001; Walsh and Groll, 1999). Although the incidence of such disease has risen in an era of aggressive chemotherapeutic regimens and stem cell transplantation (Clark and Hajjeh, 2002), our ability to detect and identify causative organisms has remained limited (O’Shaughnessy et al., 2003). Culture-based methods (Isenburg, 1998) remain the gold standard for diagnosis as they may yield the specific etiologic agent and allow for susceptibility testing to be performed (O’Shaughnessy et al., 2003). However, these methods are frequently slow

B This work was performed at St. Jude Children’s Research Hospital, Memphis, TN. BB This work was supported in part by the American Lebanese Syrian Associated Charities, Memphis, TN, USA. 4 Corresponding author: Department of Pathology, St. Jude Children’s Research Hospital, Memphis, TN 38105-2794, USA. Tel.: +1-901-4953525; fax: +1-901-495-3100. Alternate corresponding author. Chadi M. El Saleeby. Department of Pediatrics, Harvard Medical School, Massachusetts General Hospital for Children, Massachusetts General Hospital, Boston, MA 02114, USA. Tel.: +1-617-726-2553; fax: +1-617-726-5961. E-mail addresses: [email protected] (R.T. Hayden)8 [email protected] (C.M. El Saleeby).

0732-8893/$ – see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2006.04.005

and suffer from low sensitivity, particularly in the early stages of infection, when organism burden is low. Filtration techniques, initially used to assess water purity (Buck and Bubucis, 1978; Havelaar et al., 1985; Qureshi and Dutka, 1976), have been more recently applied to clinical specimens in the setting of fungal disease (Bauters et al., 1999; Bauters and Nelis, 2000; Kunin and Buesching, 2000). However, no report has evaluated the use of filters and fungal growth of cultures in comparison to growth from specimens directly applied to primary plates without filtration. We examined the utility of sample concentration by filtration-based methods to improve the sensitivity of culture for the detection of fungal organisms in clinical respiratory specimens. Seeded control specimens were created using previously identified isolates of Aspergillus fumigatus, Aspergillus flavus, Pseudallescheria boydii, and Fusarium oxysporum. Actively growing fungal isolates representing each of these control organisms were suspended in normal saline to match a 0.5-McFarland turbidity standard. Ten-fold dilutions of this stock suspension, made in normal saline, from 1:10 to 1:106 were tested. After institutional review board approval, respiratory clinical specimens from patients with cancer and patients with bone marrow transplantation as well as

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Table 1 Analytical validation of preculture filtration for A. fumigatus Dilution

0.5 Standard Control 1:10 Control Filtration 1:100 Control Filtration 1:1000 Control Filtration 1:10 000 Control Filtration 1:100 000 Control Filtration 1:1 000 000 Control Filtration

A. fumigatus colonies observed (by postinoculation day) Day 1

Day 2

Day 3

Day 4

Day 5

TNTC

TNTC

TNTC

TNTC

TNTC

TNTC TNTC

TNTC TNTC

TNTC TNTC

TNTC TNTC

TNTC TNTC

150 150

TNTC TNTC

TNTC TNTC

TNTC TNTC

TNTC TNTC

30 0

74 TNTC

74 TNTC

74 TNTC

74 TNTC

0 0

18 TNTC

24 TNTC

24 TNTC

24 TNTC

0 0

2 56

2 62

2 62

2 62

0 0

0 21

0 29

0 29

0 29

TNTC = too numerous to count.

the control specimens were tested in parallel by conventional fungal culture and by the filtration-based concentration technique. One to two drops of liquid clinical specimens as well as swab clinical specimens were inoculated to inhibitory mold agar; 0.1 mL of each dilution of seeded specimens was used as a growth control plate. The plates were streaked in 4 quadrants, shrink-sealed, incubated at 30 8C, and observed for fungal growth 3 days a week for 28 days. Any fungal growth was regarded as positive and reported in colonyforming units per milliliter. Ten milliliters of either clinical specimen or seeded control suspension was transferred to a sterile analytical filter unit. Mucoid clinical specimens were first liquefied in a sterile conical tube with an equal volume of 1% N-acetyl-l-cysteine and then incubated at 37 8C for 30 min to reduce viscosity. After vacuum filtration, the filter disk was aseptically removed and planted on appropriate fungal media with the top aspect of the filter facing down, in direct contact with the agar surface. Plates were shrink-sealed, incubated at 30 8C, and observed for fungal growth 3 days a week for 28 days. Any fungal growth was included as positive and reported in colony-forming units per milliliter. Three different filter membranes were evaluated for use in specimen concentration using seeded conidial suspensions. Membranes tested included cellulose nitrate (Nalgene Brand Products, Nagle Company, Rochester, NY), nylon (Osmonics incorporated, Minnetonka, MN), and metricel black (Pall life sciences, Ann Arbor, MI). All membrane filters were 47 mm in diameter with a pore size of 0.45 Am. The sensitivity and the rate of growth were equivalent for each filter type (results not shown). Cellulose nitrate membrane filters were available preincorporated into vacuum

filter sets. They were easier to use and were therefore chosen for further testing. McNemar’s test (Agretsi, 2002) was used to compare the detection probability for culture to that of filtration. The exact test was used to compute P values and largesample approximation to calculate 95% confidence intervals. Multiple samples were taken from some patients. Among those patients, there was a strong correlation by Fisher exact test (Agretsi, 2002) between the first 2 samples’ results by filtration ( P = 0.0030) and culture ( P = 0.0625). We attribute this correlation to the fact that consecutive samples were collected only a few days apart in several patients. Thus, in those cases, assays on consecutive samples test essentially the same condition and would likely lead to false significant findings. Therefore, McNemar’s test was applied only to the data from a set of bclinically distinct samples Q, specifically only samples collected at least 21 days apart from one another in a given patient. Twenty-one days was chosen as a threshold for inclusion in the clinically distinct set based on the assumption that clinical samples would be sterile after 3 weeks of effective antifungal therapy. We did not observe strong correlation between the first 2 clinically distinct samples in either the culture ( P = 0.2451) or filtration assays ( P = 0.6267). All samples were included in descriptive analyses. In all of the seeded conidial suspensions, filtration-based concentration method allowed detection of fungus at a 10to 100-fold lower concentration than did routine methods. However, there was no difference in time to positivity between the 2 systems. For illustrative purposes, results for seeded A. fumigatus specimens are presented in Table 1. To assess the utility of this method on clinical specimens, 186 respiratory samples were obtained from 79 patients. There were 147 endotracheal samples, 21 bronchoalveolar lavage (BAL) specimens, and 18 samples from other respiratory sites. None of the collected samples were culture swabs. There were 109 clinically distinct samples (14 BAL samples, 79 tracheal samples, and 16 other respiratory Table 2 Comparative performance of preculture filtration method and regular culture method Standard culture method

Filtration/culture method Negative

All clinically distinct cultures a Negative 76 Positive 0a Aspergillus spp. Negative Positive Candida spp. Negative Positive

Positive 9 24

108 0

1 0

77 1

8 23

Positivity difference (%) (confidence interval), P value

8.3 (3.1–13.4), 0.0039

0.9 ( 0.8 to 2.7), 1.0000

6.4 (1.2–11.7), 0.0391

Numbers in bold denote significant values. a Denotes detection of any fungal organism(s).

C.M. El Saleeby et al. / Diagnostic Microbiology and Infectious Disease 56 (2006) 221 – 223

samples). Among all clinically distinct samples, neither assay detected an organism in 76 samples, both assays detected organism in 24 samples and only filter detected organism in the remaining 9 samples (3 of which were also detected by culture from at least one other body site in the same patient). Thus, filter detected some fungal organism in 8.3% (3.1%, 13.4%) more of the clinically distinct samples than did culture ( P = 0.0039) (Table 2). Furthermore, 27.3% (9 of 33) of positive cultures were only detected using the modified filter technique. Similar analyses were conducted on individual types of organisms that grew from cultures (Table 2). Aspergillus was detected only by filter in one of the clinically distinct samples; neither assay detected Aspergillus in the remaining 108 clinically distinct samples. Candida was detected by both assays in 23 clinically distinct samples, only by filter in 8 clinically distinct samples, only by culture in one clinically distinct sample, but by neither assay in the remaining 77 samples. Thus, Candida was detected by filter in 6.4% (1.2%, 11.7%) more of the clinically distinct samples than by culture ( P = 0.0391). Among the 38 samples that were positive by both assays, culture provided a positive result before filter in 17 (44.7%) samples, filtration provided a positive result before culture in 5 (13.2%) samples, and the 2 assays reached a positive result on the same day in 16 (42.1%) samples. These differences were not statistically significant. The sensitivity of culture can be improved by this simple, rapid, and cost-effective methodology. Filtration is easy to perform and requires little or no additional training. Cellulose nitrate membrane filters are inexpensive and readily available. The use of such method produced an overall increase of 8.3% in the number of positive results, compared to routinely processed cultures. When individual organisms were analyzed, this advantage was largely related to an improved rate of detection for Candida species. The increase in sensitivity for Aspergillus spp., although showing a similar trend, did not reach statistical significance. Through the concentration of samples, filtration allowed processing of a much larger proportion of the clinical specimen submitted for testing; this may have been particularly advantageous if a sample was inhomogeneous or in cases with low organism burden, wherein partitioning may have resulted in diminished sensitivity of routine culture methods. Application of the filtration method did not significantly improve the time to positivity for clinical samples, and no meaningful difference was seen in the rate of detection of filamentous fungal isolates. This may be related to the number of samples included in this study, and the lower overall prevalence in these samples of Aspergillus and other filamentous fungal organisms, compared to Candida species. The clinical significance of increased culture sensitivity could not be determined from the available data and is thus beyond the scope of this study. However, the recovery of

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some of these organisms from nonrespiratory specimens in the same patients suggests that improved recovery may correlate in some cases to clinically useful information. Others have suggested that in high-risk populations such as ours, a positive culture result for aspergillosis from nonsterile sites is associated with invasive disease in 50% or more of cases (Perfect et al., 2001). Candida colonization has also been associated with an increased incidence of candidemia in cancer patients even in those individuals on antifungal prophylaxis (Marr et al., 2000). It is therefore not unreasonable to speculate that a test with enhanced sensitivity will detect a larger number of clinically significant isolates. Larger prospective studies are needed to evaluate the full scope of this technique and its applicability to nonrespiratory specimens.

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