Indian Journal of Medical Microbiology IAMM  | About us |  Subscription |  e-Alerts  | Feedback |  Login   
  Print this page Email this page   Small font sizeDefault font sizeIncrease font size
 Home | Ahead of Print | Current Issue | Archives | Search | Instructions  
Users Online: 323 Official Publication of Indian Association of Medical Microbiologists 
  Search
 
  
 ~  Similar in PUBMED
 ~  Search Pubmed for
 ~  Search in Google Scholar for
 ~Related articles
 ~  Article in PDF (611 KB)
 ~  Citation Manager
 ~  Access Statistics
 ~  Reader Comments
 ~  Email Alert *
 ~  Add to My List *
* Registration required (free)  

 
 ~  Abstract
 ~ Introduction
 ~  Materials And Me...
 ~ Results
 ~ Discussion
 ~  References
 ~  Article Figures
 ~  Article Tables

 Article Access Statistics
    Viewed124    
    Printed1    
    Emailed0    
    PDF Downloaded14    
    Comments [Add]    

Recommend this journal

 


 
  Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 35  |  Issue : 1  |  Page : 41-47
 

Screening of invasive fungal infections by a real-time panfungal (pan-ACF) polymerase chain reaction assay in patients with haematological malignancy


1 Department of Microbiology, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi, India
2 Department of Medicine, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi, India
3 Department of Haematology, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi, India
4 Independent Statistician, CHRD-SAS, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi, India

Date of Web Publication16-Mar-2017

Correspondence Address:
Malini Rajinder Capoor
C 99, Neelambar Apartments, Opposite Sainik Vihar, Pitampura, New Delhi - 110 034
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_15_543

Rights and Permissions

 ~ Abstract 


Background: Invasive fungal infection (IFI) is a fatal infection in haematology patients. There is an urgent need for reliable screening methods facilitating timely diagnosis and treatment. A real-time panfungal polymerase chain reaction (PCR) assay based on TaqMan technology targeting 18S ribosomal RNA gene was used to screen whole blood specimen obtained from series of Haematology malignancy patients for IFIs. Materials and Methods: The panfungal (Pan-ACF) assay was employed to investigate specimen from 133 patients in duplicate with suspected IFI. In addition twenty healthy subjects and twenty patients with bacterial infections were taken as control. The patients with suspected IFI were also diagnosed by conventional methods including direct microscopy, culture techniques and antigen detection (galactomannan antigen ELISA and latex agglutination for cryptococcal antigen). The results of molecular testing were evaluated in relation to the criteria proposed by the European Organization for Research and Treatment of Cancer and patients were classified as having proven and probable IFD. Results: Of 133 patients, 89 had proven, 18 had probable and 26 had possible IFI. One hundred four samples were reverse transcription-PCR positive. Of 89 proven cases, 84 were panfungal PCR positive. These 84 cases included 82 cases which revealed growth on fungal blood culture and two cases were negative on fungal blood culture. Of the 82 cases which revealed growth on culture: 74 grew Candida in culture, 3 grew Fusarium solani, 5 grew Aspergillus species on blood culture. The later five were also galactomannan antigen positive. The five specimen which were negative on panfungal PCR, two grew Trichosporon asahii, one grew Candida rugosa and two grew as Cryptococcus neoformans var. neoformans. Of the 18 probable cases, 18 were panfungal PCR positive. These were also galactomannan antigen positive. The sensitivity and specificity of panfungal PCR in proven cases were 94.3% and 95.2%, respectively. The positive and negative predictive values proven cases were 97.6% and 88.9%, respectively. Conclusions: The panfungal (Pan-ACF) real-time PCR assay can detect common fungal genera and it may be used as an adjunct to conventional methods for screening of IFI.


Keywords: Haematological malignancy, invasive fungal infection, panfungal real-time-polymerase chain reaction


How to cite this article:
Capoor MR, Puri S, Raheja H, Mohindra R, Gupta DK, Verma PK, Chowdhary R. Screening of invasive fungal infections by a real-time panfungal (pan-ACF) polymerase chain reaction assay in patients with haematological malignancy. Indian J Med Microbiol 2017;35:41-7

How to cite this URL:
Capoor MR, Puri S, Raheja H, Mohindra R, Gupta DK, Verma PK, Chowdhary R. Screening of invasive fungal infections by a real-time panfungal (pan-ACF) polymerase chain reaction assay in patients with haematological malignancy. Indian J Med Microbiol [serial online] 2017 [cited 2017 Mar 27];35:41-7. Available from: http://www.ijmm.org/text.asp?2017/35/1/41/202328





 ~ Introduction Top


The incidence of invasive fungal infections (IFIs) has increased in recent years due to an ever increasing the population of immunocompromised patients, especially with haematological malignancies.[1] The management of IFIs has been hindered by the inability for timely diagnosis. Atypical clinical findings, difficulty in taking samples and insufficient diagnostic methods are basic problems in patients with haematological malignancies. Expedite diagnosis is a prerequisite for successful therapy and clinical outcome in patients with invasive fungal diseases.[2],[3]

IFIs are mainly caused by Candida and Aspergillus species. These have high mortality and morbidity among immunocompromised patients.[4]Candida is the fourth most common cause of nosocomial blood stream infections. Over 200 species of Candida have been described, but 95% infections are caused by five species Candida tropicalis, Candida parapsilosis, Candida glabrata, Candida krusei and Candida albicans. Invasive aspergillosis is mainly caused by Aspergillus fumigatus and Aspergillus flavus. Other species associated are Aspergillus nidulans, Aspergillus terreus, Aspergillus niger. Other pathogen causing fungaemia are Cryptococcus, fusarium, Scedosporium, Mucorales and others.[5]

The standard methods for the detection of fungal infections are direct microscopy and culture. Blood for fungal cultures are positive for fewer than 50% of patients with hepatosplenic candidiasis and are rarely positive for patients with invasive aspergillosis. Even cultures of bronchoalevolar lavage are frequently negative. Histological analysis of computer tomography-guided biopsies are highly sensitive and specific, but are usually associated with bleeding complications in patients with severe thrombocytopenia.[5],[6],[7] Rapid diagnostic methods for fungal infections include the detection of antibody, serological detection of circulating fungal antigen (e.g., D-glucan or galactomannan) and DNA. These have shown variable sensitivity and specificity. Furthermore, immunocompromised patients have poor antibody response.[8]

Recently, a variety of polymerase chain reaction (PCR)-based methods have been developed for rapid and sensitive detection of fungal pathogens.[9] A few studies by daily blood sampling have revealed in many cases fungal PCR was positive several days before fungal pathogens like Candida could be detected by blood culture.[10] A considerable amount of published assays only permit detection of single fungal species or few Candida[2],[10],[11] or Aspergillus species in blood samples of patients with IFIs.[4],[7],[8] Furthermore, the clinical utility of a species specific or a genus specific PCR assay is limited.[7] In literature, there are few reports of Pan-AC (detecting Aspergillus and Candida spp. both)[4],[6] and assays targeting multiple fungal genera.[3],[5] The studies depicting analytical ability of real-time panfungal PCR to detect IFIs as per the European Organization for Research and Treatment of Cancer (EORTC) guidelines [12],[13],[14] are even fewer. The aim of this study was to an evaluate a commercial PCR assay and to show whether panfungal real-time PCR assay could be used as an adjunct to conventional methods for screening and expedient diagnosis of IFIs.


 ~ Materials And Methods Top


During the prospective study (2011–2012), a total of 133 clinical blood samples in duplicate were analysed. Samples were obtained on admission from hospitalised patients naïve to antifungals with haematological malignancy with fever and with suspected IFI from Haematology ward. These were classified into proven, probable and possible as per the EORTC criteria later. In addition, blood from healthy controls (20) and patients suffering from bacterial sepsis (20) were included in the study.

In brief, the haematological malignancy patients with suspected IFD had clinical symptoms and characteristics included: Host factors with persistent fever, unresponsiveness to broad spectrum antibacterial therapy, colonisation with multiple sites, repeated computed tomography and ultrasound scan suggesting a mycotic lesion. For proven IFI, the mycological criteria included isolation of fungi from blood or other sterile sites and specimen positivity by histopathology. For probable IFI, the mycological criteria included antigen positivity (galactomannan antigen), isolation of fungi by direct microscopy and culture from non-sterile sites. Possible IFD included cases with appropriate host factors and clinical evidence consistent with IFD for which there was no mycological support. Sample collection: Whole blood (5 ml) samples were collected in ethylenediaminetetraacetic acid vacutainer for PCR assay, before starting antifungal therapy. Whole blood (5 ml) in plain vacutainer was collected for serum galactomannan antigen detection (Bio-Rad, California, USA). In addition, latex agglutination tests for cryptococcal antigen were also carried out on patients serum and cerebrospinal (CSF) sample, wherever indicated. All blood samples were stored at − 80°C until DNA extraction.

Depending on the time and infection, additional samples derived from primary sterile sites (blood, CSF, bone marrow, etc.) of suspected infection and non-sterile sites (endotracheal aspirate, urine, wound site and sputum) to differentiate infection from colonisation were also collected. These were processed for fungal culture to correlate the data with PCR. Whole blood samples were collected concurrently with those taken for bacterial and fungal culture before initiation of antifungal therapy. All the standardised precautions for molecular biology laboratory were taken during reactions.

Polymerase chain reaction validation

Reference fungal strains: The ability of the panfungal system to detect all fungus species of interest was determined by testing DNA derived from cultures of reference strains, including C. tropicalis (ATCC 750), C. albicans (ATCC 14053), Candida dubliniensis (ATCC MYA-646), C. glabrata (ATCC 2001), C. krusei (ATCC 6258), C. parapsilosis (ATCC 22019), Candida guilliermondii (DSM 70051), Candida kefyr (DSM 70073), Candida lusitaniae (DSM 70102), Aspergillus versicolor (DSM 1943), A. fumigatus (ATCC 36607), A. niger (ATCC 10535), A. terreus (DSM 826), Fusarium solani.

Fungal strains for PCR testing were obtained from the American Type Culture Collection (ATCC) and from the German Collection of Micro-organisms (DSM, Germany). The DNA extracted from C. neoformans, Candida rugosa, A. glaucus gave a negative panfungal PCR result.

Quality control

Sensitivity and reproducibility

For sensitivity of the panfungal real-time PCR kit, a dilution series was setup from106 down to 10 copies/µl of panfungal DNA and analysed with the panfungal real-time PCR kit. The assays were carried out on three different days in the form of 8-fold determinations. The detection limit as determined for kit was consistently 50 copies/ml (for all the included species: Candida spp., Aspergillus spp., Fusarium spp.).

Specificity

Different DNA were analysed (Eschericha coli, Proteus vulgaris, Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae, Mycobacterium tuberculos is). None of these led to a positive signal with the panfungal real-time PCR reaction. Twenty healthy volunteers and patients suffering from bacterial sepsis (20) were panfungal PCR negative (negative control).

The definition of IFI was based on the 2008 EORTC/MSG guidelines.[12] Decisions about whether patients had proven, probable or possible IFI were made strictly independent from PCR results. The PCR results were evaluated by correlating them with the clinical classification. Results of microbiological tests of culture (sterile and non-sterile sites) and serology were included for classifications.

DNA extraction

The DNA extraction of the blood sample, ATCC fungal and bacterial strains was performed using the Qiagen QIAamp DNA Mini Extraction Kit as per the manufacturer's instructions. For effective lysis of fungal cell wall heat shock method was incorporated additionally in the Qiagen protocol. After DNA extraction, spectral photometry was done to measure DNA purity and content, wherever applicable.

For the assay a commercial kit (Geno-Sen's panfungal real-time PCR kit, Genome Diagnostics Private Limited, India) was used on Rotor Gene™ 6000, Corbett Research, Australia (real- time PCR instrument). Primers for Aspergillus spp. and Candida spp. as reported previously [6] and in addition to these primers for Fusarium spp. were used to amplify the judiciously selected region of the 18S subunit of the fungal ribosomal DNA gene (GenBank accession no: EF093258.1), common to clinically relevant six Aspergillus, nine Candida and one Fusarium spp. were selected and aligned using BLAST search software (accession no JX515972.1, JX233485.1, JX2204287.1, JM 391347.1 for Aspergillus and Candida spp. and accession no. JX 244004.1 for Fusarium spp.), a highly conserved region of 18S rDNA was identified (GenBank Accession no: EF093258.1), optimally fitting the requirements of PCR analysis using a hydrolysis TaqMan probe. The probe was labelled with 6-carboxyfluorescein as a reporter molecule at the 5'-end and BHQ as a quencher molecule at the 3'-end. The optimal concentrations for the primers and the probe were assessed by serial analyses both from the functional and economic perspective.

Molecular analysis

Principle of real-time PCR: The robust assay exploits TaqMan principle targeting 18S ribosomal RNA gene (GenBank accession no: EF093258.1). During PCR, forward and reverse primers hybridise to a specific sequence product. A TaqMan probe, which is contained in the same reaction mixture and which consists of an oligonucleotide labelled with a 5'-reporter dye and a downstream, 3'-quencher dye, hybridises to a target sequence within the PCR product. A Taq polymerase which possesses 5'-3' exonuclease activity cleaves the probe. The reporter dye and quencher dye are separated on cleavage, resulting in an increase in fluorescence for the reporter. Thus, the increase in fluorescence is directly proportional to the target amplification during PCR.

Amplification was performed using the following protocol: hold for 10 min at 95°C (activation of TaqDNA polymerase), for 10 min. Followed by denaturation step 95°C for 15 s. Setting up of Annealing step in the cycling profile as 55°C for 20 s and Extension step in the cycling profile as depicted below, that is, 72°C for 15 s (45 cycles in the cycling profile as target amplification) and 30 s at 72°C.

Definition of polymerase chain reaction positivity and DNAemia

All clinical specimens were analysed in duplicates. For the diagnosis of fungal DNAemia, a minimum of two PCR-positive peripheral blood specimens derived at subsequent time points during close follow-up investigation were required. In the rare instances in which only one specimen from a febrile neutropenic episode was available, a single PCR positive result was considered as indicative of DNAemia.

All PCR runs included positive and negative controls of kit and an in house positive control (containing DNA of one of fungal isolates) and a negative control (water in place of DNA). All the PCR reactions were performed as per Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines.[15]

Statistical analysis

Results of panfungal PCR analysis in relation to the presence or absence of possible, probable or proven IFD by the EORTC criteria were used for the calculation of sensitivity and specificity, and the corresponding negative predictive values (NPV) and positive predictive values (PPVs) of the assay.


 ~ Results Top


A total of 133 patients with haematological malignancy were included in the study. The age of the patients ranged from 8 to 66 years (median 21 years). The spectrum of underlying haematological malignancy with febrile neutropenia was acute lymphoblastic leukaemia (52), acute myeloid leukaemia (39), chronic myeloid leukaemia (32), chronic lymphoblastic leukaemia (3) and others (7). All these patients were naïve to antifungals. Of 133 patients, 89 had proven, 18 had probable and 26 had possible IFI. One thirty-three whole blood samples from 133 patients were processed in duplicate. One hundred four samples were reverse transcription-PCR (RT-PCR) positive. Of 133 patients, 89 had proven, 18 had probable and 26 had possible IFI. [Table 1] depicts PCR results with different patient groups of IFI as per EORTC/MSG 2008 criteria.
Table 1: Polymerase chain reaction results with different patient groups of invasive fungal infections as per the European Organization for Research and Treatment of Cancer/Mycoses Study Group 2008 criteria

Click here to view


Of 89 proven cases, 84 were panfungal PCR positive. [Table 2] depicts performance of real-time PCR in proven (89) and control (40) cases. These 84 cases included 82 cases which revealed growth on fungal blood culture and two cases were negative on fungal blood culture. Of the 82 cases which revealed growth on culture: 74 grew Candida in culture, 3 grew F. solani, 5 grew Aspergillus species on blood culture [Table 3]. The later five were also galactomannan antigen positive. The five specimen which were negative on panfungal PCR, two grew Trichosporon asahii, one grew C. rugosa, and two grew as C. neoformans var. neoformans.
Table 2: Performance of real-time polymerase chain reaction in proven (89) and control (40) cases

Click here to view
Table 3: Distribution of various fungal isolates in the study which were pan fungal polymerase chain reaction positive

Click here to view


Of the 18 probable cases, 18 were panfungal PCR positive. These were also galactomannan antigen positive. The sensitivity and specificity of panfungal PCR in proven cases were 94.3% and 95.2%, respectively. The PPV and NPV proven cases was 97.6% and 88.9%, respectively. In probable cases sensitivity, specificity, PPV and NPV of panfungal PCR was 100% each, respectively.

Only 2 out of 26 cases of possible IFD from febrile neutropenia were on antifungal was panfungal PCR positive. Blood from healthy controls (20) and patients suffering from bacterial sepsis (20) was panfungal PCR negative. [Table 4] shows quantitation information of a run of cases samples and controls in the study. [Figure 1] shows quantitation data of samples from cases, standards and control for Cycling A. Green. [Figure 2] depicts standard curve of the cases samples and controls. Panfungal PCR assay showing the results of positive control standards (S1, S2, S3, S4), negative control (NTC) and patients cases sample (sample 3, 4, 7, 8, 9) [Table 5].
Table 4: Quantitation information of the representative samples in the study

Click here to view
Figure 1: Quantitation data of samples, standards and control for Cycling A.Green.

Click here to view
Figure 2: Standard curve of the representative samples.

Click here to view
Table 5: Pan fungal polymerase chain reaction assay showing the results of positive control standards (S1, S2, S3, S4), negative control (NTC) and patients sample (sample 3, 4, 7, 8, 9)

Click here to view


The authors assessed the validity and reliability of panfungal PCR in relation to proven cases, therefore only sensitivity, specificity, NPV and PPV value and their 95% confidence intervals was calculated. The authors used MedCalc software to assess the diagnostic test panfungal PCR. As we did not assess two different diagnostic PCRs/tests in comparison to proven IFIs so there was no need to calculate statistical significance.


 ~ Discussion Top


In this study protocol, 18S rRNA was targeted, since this region has conserved sequences in multiple copies, whereas in prior studies others such as ITS1 and ITS2, mitochondrial DNA was targeted, which is highly variable. To increase the detection and quantitative monitoring of pathogenic fungi in clinical samples the real-time PCR was used. The development of real-time PCR has largely replaced block-based PCR diagnosis and provides greater sensitivity, specificity, speed, convenience and accuracy. Its technology combines rapid in vitro amplification of DNA with real-time detection and quantification of target molecules present in samples.[16],[17] Currently, there are protean range of PCR assays utilising specific bi-probes, hydrolysis (TaqMan) and hybridisation (fluorescence resonance energy transfer) probes in literature.[9], 15, [16],[17],[18],[19],[20] In this study, we used the TaqMan probe. The analytical sensitivity of fungal RT-PCR was 50 copies/ml. This is definitely an improvement from PCR with 100 copies/ml. In this study, primers were found is to be highly specific as their ability to identify their true target organism. As the fungal burden is limited in blood samples so the detection limit needs to be at a level that almost certainly does not compromises specificity.[9]

For proven IFI sensitivity and specificity of the panfungal PCR assay was 94.3% and 95.2%, respectively with a PPV of 97.6% and NPV of 88.9%. The sensitivity and specificity is comparable to an Austrian study performed for the diagnosis of IFI by a real- time panfungal PCR assay on immunocompromised (neutropenic) patients.[3] Our result is also comparable to an Indian study [4] where in 100% sensitivity and specificity with a PPV of 100% and a NPV of 96.7% was observed for panfungal nested PCR. However, our study showed higher sensitivity, specificity, PPV and NPV compared to a recent Indian study,[13] on prospective screening of Pan-AC assay targeting 28S rRNA gene in paediatric leukaemia patients, where in the respective percentages were very low (50%, 36%, 11%, 81%, respectively). This was attributed to low number of patients with possible (4), probable (0) and proven (1) IFIs. The authors also commented that the drawback in their study was low volume of peripheral blood (200 ml) as compared to the recommended guideline of 3 ml.[13],[17],[18] A prior study from Egypt the respective percentages were comparatively lower (75%, 92%, 84% and 87%, respectively). The authors attributed this to conventional PCR and use of serum as sample.[18] A prior similar study on proven cases included 34 patients (haematological and non-haematological cases), exposed to antifungal therapy before biopsy sample collection. On the patient level, the panfungal PCR revealed a sensitivity, specificity, PPV and NPV of 100%, 62.5%, 83% and 100%, respectively. Compared with this study our panfungal PCR had a comparable sensitivity, the specificity was higher and NPV and PPV was in the range of previously published data.[21],[22] Reasons for differences in specificity might be the choice of 'goldstandard' for comparison: Most studies calculated sensitivity, specificity, PPV and NPV of their panfungal PCR approach using all three EORTC categories (proven, probable and possible)[3] or two of these (proven and probable) IFI together.[20]

The sensitivity and NPV of an panfungal assay was lesser as compared to a few prior studies,[3] as this panfungal PCR assay was specific to common species of fungi like six species of Aspergillus, eight species of Candida and one species Fusarium. In five patients, the panfungal PCR was negative, but the blood culture sample from the same patients grew T. asahii (2), C. rugosa (1) and 2 C. neoformans var. neoformans, respectively. Several possible explanations for this false-negative signal include sampling error, prolonged cold storage time, transposition of specimen tubes, or suboptimal yield from the DNA extraction.[17]

The Austrian study targeted multiple fungal genera, thereby increasing the cost of the test per patient.[3] The sensitivity and NPV of panfungal PCR assay can be increased by using primers targeting the multiple fungal genera. However, this would decrease the cost effectiveness of the kit. In limited resource setting, a balance between sensitivity, NPV and cost effectiveness of the test have to be maintained. As a perfect PCR system generates ultimate sensitivity without compromising specificity and it aids to rule in or exclude infection. A highly sensitive assay will yield false positive results. Therefore a balance between the early detection of subclinical infection and contamination is also crucial.[9] Real-time PCR assay using the LightCycler technique combines rapid amplification, probe hybridisation and signal generation in a single generation in a single tube, allowing quantification of fungal DNA while retaining sensitivity.[18],[23] Other factors attributed to increase in sensitivity of PCR was to obtain sample before AFT and serial sampling. In this study from suspected IFI cases, samples were collected before initiation of AFT. As the antifungal therapy reduces the fungal load within the host and it alters the fungal target by damaging the fungal cell wall and membrane, releasing the DNA and providing a pre-circulating DNA rather than cell associated DNA source and it may generate a false negative result,[19] as the methodologies for DNA purification are different for circulating and cell-associated DNA.[9]

The best accuracy in probable cases was more than that in proven cases, this could have been a coincidence as the number of probable cases were much less than proven cases. Other reason attributed could be, as all the probable cases were of invasive aspergillosis, as this panfungal PCR assay was specific to six most common species of Aspergillus causing invasive aspergillosis. In this study, false positive PCR result was seen in two cases of proven IFI and two cases of possible fungal infection. This is linked to possible contamination entering during multistep fungal DNA extraction procedure. In this study, the contamination was controlled by use of standard molecular biology precautions and use of certain reagents such as zymolase, was avoided. As this assay utilises panfungal oligonucleotides targeting the 18S rRNA complex, the conserved regions may also would have shared significant similarity with human homologous gene complex and would have given a false positive or false negative result.[9],[17],[18],[19] Furthermore, it has been suggested that inclusion of multiple negative controls in each run (one control for every five samples) avoids ambiguous results.[17] None the less, the use multicopy genes (18S, 28S, D1, D2, ITS, etc.) have better sensitivity than single copy genes (beta-actin, alkaline phosphatase, histone, etc.), it is well documented in literature, especially for panfungal assays.[3],[9],[17]

Only in one case out of 26 possible cases of IFD from febrile neutropenics was panfungal PCR positive. This indicates that almost 90% of febrile neutropenic episodes would have been treated empirically by AFT as is routine in Haematology patients, despite the lack of microbiological and radiological evidence for IFD as per the EORTC criteria. This is suggestive of the fact that a high population of these episodes in febrile neutropenic patients would have been over treated. Therefore, molecular test may perhaps be useful in preventing unnecessary treatment, as was seen in this study.

The heterogenecity of study populations, specimen, target genes, methods of DNA extraction, treatments, designs of PCR amplification and disease definitions makes direct comparison difficult. Nonetheless, optimal PCR methodologies may be combined with an adequate DNA extraction procedure on an appropriate specimen. Furthermore, some form of standardisation is required to make an international comparison possible. Any cost effective panfungal PCR should detect the most frequent and prevalent Candida species (C. tropicalis, C. parapsilosis, C. albicans) and should be also able to detect possibly azole resistant species (C. krusei and C. glabrata), as was seen in this study. Furthermore, the assay in this study targeted the pan-ACF species (Aspergillus, Candida, Fusarium), targeting the most common species causing IFI in haematology patients. However, there are few reports documenting use of this kind in ocular samples using nested PCR.[24],[25]

There are studies in literature dealing with panfungal PCR assays allowing exact identification of the fungus by the use of sequencing for post-amplification identification.[1],[3],[7],[9] However, post-amplification sequencing too slow, labour intensive and expensive for use diagnostically and its successful application may be reliant on DNA extracted from culture organisms rather than specimen testing, standardised PCR is preferred over sequencing.[9] Recently, FungiQuant, a broad-coverage fungal quantitative and detection tool using real-time PCR technology followed by primer-probe sequence sillica analysis was developed for diverse fungi.[16]

The combined use of newer modalities for the diagnosis of IFIs in haematological patients such as galactomannan, 1,3-β-D-glucan, Aspergillus PCR, multifungal DNA-microarray, and Aspergillus azole resistance PCRs in blood and bronchoalveolar lavage samples ensures higher sensitivity, specificity, NPV and PPV of diagnostic tests for better patient management.[26]

The molecular screening by real-time panfungal (Pan-ACF) PCR is rapid, reliable and cost effective way for identification of fungaemia in haematology patients. If standardised properly PCR results can be incorporated in consensus criteria in probable category and may be used with additional microbiological and clinical information. Furthermore, multi-centre clinical evaluation using a standard protocol is warranted for PCR to be incorporated in consensus criteria. In addition, routine employment of a broad set of diagnostic methods as per EORTC criteria using various modalities of the computed tomography, mycological tests, fungal blood cultures and other serological testing is required to use the panfungal PCR screening assay to the best of its diagnostic potential.

The panfungal (Pan-ACF) assay provides an attractive and economic approach to the screening and monitoring of invasive aspergillosis and candidiasis, which is readily applicable to routine clinical diagnosis, especially in the resource constrain setting where cost-effective molecular assay is warranted. Furthermore, it also proved superior to culture in early diagnosis of IFIs in patients with febrile neutropenia. Dependence on conventional tests alone in early stage would result in misdiagnosis and its consequent delayed management in patients with haematological malignancies.

Acknowledgement

The authors would like to thank Late Mrs. Kamlawati, Senior Technician, Mycology, Department of Microbiology, V.M.M.C. and Safdarjung Hospital, New Delhi, for processing of samples for fungal culture.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 ~ References Top

1.
Van Burik JA, Myerson D, Schreckhise RW, Bowden RA. Panfungal PCR assay for detection of fungal infection in human blood specimens. J Clin Microbiol 1998;36:1169-75.  Back to cited text no. 1
    
2.
Maaroufi Y, Ahariz N, Husson M, Crokaert F. Comparison of different methods of isolation of DNA of commonly encountered Candida species and its quantitation by using a real-time PCR-based assay. J Clin Microbiol 2004;42:3159-63.  Back to cited text no. 2
    
3.
Landlinger C, Preuner S, Bašková L, van Grotel M, Hartwig NG, Dworzak M, et al. Diagnosis of invasive fungal infections by a real-time panfungal PCR assay in immunocompromised pediatric patients. Leukemia 2010;24:2032-8.  Back to cited text no. 3
    
4.
Deshpande P, Shetty A, Mehta A, Kapadia F, Hedge A, Soman R, et al. Standardization of fungal polymerase chain reaction for the early diagnosis of invasive fungal infection. Indian J Med Microbiol 2011;29:406-10.  Back to cited text no. 4
[PUBMED]  [Full text]  
5.
Vollmer T, Störmer M, Kleesiek K, Dreier J. Evaluation of novel broad-range real-time PCR assay for rapid detection of human pathogenic fungi in various clinical specimens. J Clin Microbiol 2008;46:1919-26.  Back to cited text no. 5
    
6.
Basková L, Landlinger C, Preuner S, Lion T. The pan-AC assay: A single-reaction real-time PCR test for quantitative detection of a broad range of Aspergillus and Candida species. J Med Microbiol 2007;56(Pt 9):1167-73.  Back to cited text no. 6
    
7.
Lau A, Chen S, Sorrell T, Carter D, Malik R, Martin P, et al. Development and clinical application of a panfungal PCR assay to detect and identify fungal DNA in tissue specimens. J Clin Microbiol 2007;45:380-5.  Back to cited text no. 7
    
8.
Hummel M, Spiess B, Roder J, von Komorowski G, Dürken M, Kentouche K, et al. Detection of Aspergillus DNA by a nested PCR assay is able to improve the diagnosis of invasive aspergillosis in paediatric patients. J Med Microbiol 2009;58(Pt 10):1291-7.  Back to cited text no. 8
    
9.
White PL, Perry MD, Barnes RA. An update on the molecular diagnosis of invasive fungal disease. FEMS Microbiol Lett 2009;296:1-10.  Back to cited text no. 9
    
10.
Tirodker UH, Nataro JP, Smith S, LasCasas L, Fairchild KD. Detection of fungemia by polymerase chain reaction in critically ill neonates and children. J Perinatol 2003;23:117-22.  Back to cited text no. 10
    
11.
Bennett J. Is real-time polymerase chain reaction ready for real use in detecting candidemia? Clin Infect Dis 2008;46:897-8.  Back to cited text no. 11
    
12.
De Pauw B, Walsh TJ, Donnelly JP, Stevens DA, Edwards JE, Calandra T, et al. Revised definitions of invasive fungal disease from the European Organization 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) Consensus Group. Clin Infect Dis 2008;46:1813-21.  Back to cited text no. 12
    
13.
Mandhaniya S, Iqbal S, Sharawat SK, Xess I, Bakhshi S. Diagnosis of invasive fungal infections using real-time PCR assay in paediatric acute leukaemia induction. Mycoses 2012;55:372-9.  Back to cited text no. 13
    
14.
Lass-Flörl C, Mutschlechner W, Aigner M, Grif K, Marth C, Girschikofsky M, et al. Utility of PCR in diagnosis of invasive fungal infections: Real-life data from a multicenter study. J Clin Microbiol 2013;51:863-8.  Back to cited text no. 14
    
15.
Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clin Chem 2009;55:611-22.  Back to cited text no. 15
    
16.
Liu CM, Kachur S, Dwan MG, Abraham AG, Aziz M, Hsueh PR, et al. FungiQuant: A broad-coverage fungal quantitative real-time PCR assay. BMC Microbiol 2012;12:255.  Back to cited text no. 16
    
17.
Isik N, Saunders NA. Application of Real-Time PCR to the Diagnosis of Invasive Fungal Infection. Current PCR. Available from: http://www.horizonpress.com/pcrbooks. [Last accessed on 2016 Feb 24].  Back to cited text no. 17
    
18.
White PL, Barnes RA. Aspergillus PCR. In: Latge JP, Steinbach W, editors. Aspergillus fumigatus and Aspergillosis. Ch. 39. Washington, DC: ASM Press; 2008. p. 373-90.  Back to cited text no. 18
    
19.
White PL, Perry MD, Loeffler J, Melchers W, Klingspor L, Bretagne S, et al. Critical stages of extracting DNA from Aspergillus fumigatus in whole-blood specimens. J Clin Microbiol 2010;48:3753-5.  Back to cited text no. 19
    
20.
El-Mahallawy HA, Shaker HH, Ali Helmy H, Mostafa T, Razak Abo-Sedah A. Evaluation of pan-fungal PCR assay and Aspergillus antigen detection in the diagnosis of invasive fungal infections in high risk paediatric cancer patients. Med Mycol 2006;44:733-9.  Back to cited text no. 20
    
21.
Babouee B, Goldenberger D, Elzi L, Lardinois D, Sadowski-Cron C, Bubendorf L, et al. Prospective study of a panfungal PCR assay followed by sequencing, for the detection of fungal DNA in normally sterile specimens in a clinical setting: A complementary tool in the diagnosis of invasive fungal disease? Clin Microbiol Infect 2013;19:E354-7.  Back to cited text no. 21
    
22.
Gupta P, Ahmad A, Khare V, Kumar A, Banerjee G, Verma N, et al. Comparative evaluation of pan-fungal real-time PCR, galactomannan and (1-3)-ß-D-glucan assay for invasive fungal infection in paediatric cancer patients. Mycoses 2016; doi:10.1111/myc.12584 [Epub ahead of print].  Back to cited text no. 22
    
23.
Loeffler J, Henke N, Hebart H, Schmidt D, Hagmeyer L, Schumacher U, et al. Quantification of fungal DNA by using fluorescence resonance energy transfer and the light cycler system. J Clin Microbiol 2000;38:586-90.  Back to cited text no. 23
    
24.
Jaeger EE, Carroll NM, Choudhury S, Dunlop AA, Towler HM, Matheson MM, et al. Rapid detection and identification of Candida, Aspergillus, and Fusarium species in ocular samples using nested PCR. J Clin Microbiol 2000;38:2902-8.  Back to cited text no. 24
    
25.
Bagyalakshmi R, Therese KL, Madhavan HN. Application of semi-nested polymerase chain reaction targeting internal transcribed spacer region for rapid detection of panfungal genome directly from ocular specimens. Indian J Ophthalmol 2007;55:261-5.  Back to cited text no. 25
[PUBMED]  [Full text]  
26.
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-ß-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.  Back to cited text no. 26
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

Top
Print this article  Email this article
 

    

2004 - Indian Journal of Medical Microbiology
Published by Wolters Kluwer - Medknow

Online since April 2001, new site since 1st August '04