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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 42  |  Issue : 4  |  Page : 142-147

Diagnosis of fungemia among pediatric patients with hematological malignancies: value of panfungal polymerase chain reaction


1 Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt
2 Department of Pediatrics, Faculty of Medicine, Ain Shams University, Cairo, Egypt

Date of Submission05-Sep-2017
Date of Acceptance02-Oct-2017
Date of Web Publication9-Feb-2018

Correspondence Address:
Marwa A El-Ashry
Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ejh.ejh_41_17

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  Abstract 


Introduction Invasive fungal infections (IFIs) remain a significant threat to pediatric patients with hematological malignancies and to those undergoing stem cell transplantation. IFIs not only increase mortality and morbidity but may also lead to delayed administration of chemotherapy, prolonged hospitalization, and additional costs associated with antifungal therapy. Early detection and appropriate treatment is crucial for the survival of these patients.
Aim The aim was to determine the value of real-time (RT) PCR, using a panfungal marker, in screening pediatric patients with hematological malignancies for early detection of IFIs.
Patients and methods This study included 50 children previously diagnosed with different hematological malignancies and admitted to the Pediatric Hematology, Oncology and Intensive Care Units of Ain Shams University Children Hospital for treatment and follow-up. Children were clinically suspected as having an IFI. There were 20 (40%) females and 30 (60%) males, with their ages ranging from 2 to 18 years (median: 6; interquartile range: 4–10.25). Venous blood was collected from all patients and was submitted for the diagnosis of IFI by conventional blood culture and RT-PCR assay using universal fungal primers for amplification of the internal transcribed spacer 1 and internal transcribed spacer 4 regions.
Results Of the 50 studied cases, 40% were positive for IFI by both blood culture and the panfungal PCR. A total of 12 (24%) patients were positive by the panfungal PCR only and the remaining 36% were negative by both assays. There was a statistically moderate agreement between the results of blood culture and that of RT-PCR for the detection of the panfungal marker (κ=0.545).
Conclusion RT-PCR assay, using the panfungal marker, is a rapid and sensitive assay that can be reliably used for screening hematological malignant patients at high risk of IFIs. The negative predictive value of the assay is 100%; thus, it can provide greater confidence in excluding a diagnosis of IFIs when negative results are obtained. This, in turn, can help prevent unnecessary toxicity resulting from empirical antifungal treatment in individuals who may not be at risk of imminent fungal disease. However, the RT-PCR for the detection of the panfungal marker lacks specificity, making the interpretation of positive results a challenge.

Keywords: Candida albicans, invasive fungal infection, panfungal marker, real-time polymerase chain reaction


How to cite this article:
El-Ashry MA, Ragab EA. Diagnosis of fungemia among pediatric patients with hematological malignancies: value of panfungal polymerase chain reaction. Egypt J Haematol 2017;42:142-7

How to cite this URL:
El-Ashry MA, Ragab EA. Diagnosis of fungemia among pediatric patients with hematological malignancies: value of panfungal polymerase chain reaction. Egypt J Haematol [serial online] 2017 [cited 2018 Jul 23];42:142-7. Available from: http://www.ehj.eg.net/text.asp?2017/42/4/142/225091




  Introduction Top


Invasive fungal infections (IFIs) are considered among the most important causes of morbidity and mortality in pediatric patients with hematological malignancies. Several factors such as neutropenia (especially for acute myeloid leukemia), damaged mucosa, excessive usage of steroids, high-dose chemotherapy, invasive medical procedures, genetic profile, and broad-spectrum antibacterial drugs usage constitute the probable risk factors for IFIs [1].

Among children with hematological malignancies, the effect of IFIs can be devastating, with a high rate of mortality and morbidity despite the development of more active, less toxic antifungal agents, and the use of antifungal prophylaxis [2]. Thus, expedited diagnosis and initiation of appropriate antifungal therapy are a prerequisite for successful therapy and clinical outcome in patients with IFIs [3].

Although the actual incidence of the IFIs has increased, its real frequency is often underestimated because of the difficulty in securing a firm diagnosis as most patients may not exhibit specific signs and symptoms related to IFIs. The traditional microbiological workup of clinical specimens is based on microscopic examination, culture on various media, and serological tests [4]. However, no method has been proven sufficiently sensitive and specific to allow adequate diagnosis, and the ‘gold standard’ consists of microscopy and culture. Atypical clinical findings, difficulty in taking samples, and insufficient diagnostic methods are basic problems in patients with hematological malignancies [5].

Over the past 2 decades, molecular techniques have been implemented for accurate pathogen identification in diagnostic microbiology [6]. Broad-range internal transcribed spacer (ITS) rRNA gene PCR is used to detect and successfully identify fungal pathogens predominantly in immunosuppressed patients, as it offers the potentiality of being more sensitive than current culture-based methods, encompassing multiple fungal genera, and being applied to a variety of specimen types [7].

Compared with conventional PCR, the introduction of real-time (RT) PCR has increased the reliability of the results. Moreover, RT-PCR can give the results in less than 2 h, which is an important requirement for clinical decision making [8].

The present study was aimed to determine the value of RT-PCR, using a panfungal marker, in screening pediatric patients with hematological malignancies for early detection of IFIs.


  Patients and methods Top


The study was conducted at the Central Microbiology Laboratory, Clinical Pathology Department at our University Hospital, over the period between November 2013 and August 2014.

Study population

The study included 50 patients who were attending the Pediatric Hematology and Oncology Unit of our Hospital (44/50, 88% diagnosed as having acute lymphoblastic leukemia and 6/50, 12% diagnosed as having acute myeloid leukemia). All studied children were experiencing fever not responding to broad-spectrum antibiotics for at least 48 h and had at least one host factor suggestive of IFI according to the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group (EORTC) and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (MSG) classification. The definitions assigned three levels of probability to the diagnosis of IFIs that develop in immunocompromised patients, namely, ‘proven’, ‘probable’, and ‘possible’ IFIs [3]. There were 20 (40%) female and 30 (60%) male patients, with ages ranging from 2 to 18 years (median: 6; interquartile range: 4–10.2).

Full history was taken from all patients with special emphasis on symptoms suggestive of local and/or systemic infection, history of antibiotic or antifungal administration, history of chemotherapy, and the duration of the disease.

Specimen collection

Venous blood was collected from each patient under aseptic condition. Collected blood was distributed as follows:
  1. A volume of 1–3 ml was injected into a BACTEC Peds Plus (Becton, Dickinson and Company (BD), Franklin Lakes, New Jersey, USA) blood culture bottle.
  2. A volume of 3 ml was mixed with EDTA and was recruited for panfungal PCR.
  3. A volume of 2 ml was injected into another EDTA-containing tube and was submitted for complete and differential blood count using Beckman–Coulter (GENs; California, USA.).
  4. A volume of 2 ml was injected into a plain tube for the assessment of C-reactive protein (CRP) using CRP-latex slide agglutination test (Spinreact, Girona, Spain).


Specimens processing

Blood culture for fungus isolation

BACTEC Ped Plus bottles were inserted into the BACTEC FX40 instrument, where bottles are screened every 10 min for the presence of fluorescence signals indicative of active microbial replication. Overall, 0.5 ml blood–broth mixture was aspirated from the positive blood culture vial and subcultured onto two plates of Sabouraud dextrose agar (Oxoid Company, UK) for the isolation of moulds and yeasts. One plate was incubated at 36±1°C, and the other was incubated at 27±1°C under aerobic conditions. Plates were inspected every other day for fungal growth for 2 weeks and then twice weekly for the next 2 weeks. Growth was identified by colonial morphology and microscopic appearance. Yeasts were further identified to the species level using the VITEK 2 Compact YST identification card (BioMerieux, Marcy-l’Étoile, France).

Real-time polymerase chain reaction using panfungal marker

Genomic DNA was extracted using Thermo Scientific GeneJET Whole Blood Genomic DNA Purification Mini Kit (St. Louis, Missouri USA) according to manufacturer’s instructions. Amplification of the extracted DNA was carried out using Maxima SYBR Green qPCR Master Mix (2×) (Waltham, Massachusetts, USA). A universal fungal primer for amplification of the ITS1 and ITS4 regions was used. The primer sequences were as follows: forward primer 999: 5′-GATACCGTCGTAGTCTTA-3′ and reverse primer 1574c: 5′-ATTCCTCGTTGAAGAGC-3′ [9]. The sequence of the forward primer ITS1 is complementary to a conserved region at the end of the 18s rRNA gene, and the sequence of the reverse primer ITS4 binds to a conserved region at the beginning of the 28s rRNA gene, leading to amplification of the ITS regions and the 5.8s rRNA gene, which is located between the noncoding ITS regions [10].

Thermal cycler was programmed under the following cycling conditions: 15 min of initial denaturation at 94°C, followed by 40 cycles of 30 s denaturation at 94°C, 90 s of annealing at 60°C, and 90 s of extension at 72°C. At the end of 40 cycles, a 10 min extension was performed at 72°C.

Statistical analysis

The data were analyzed using IBM SPSS advanced statistics (version 23.0, 2015; SPSS Inc., Chicago, Illinois, USA). Data were expressed as median and percentiles for quantitative nonparametric measures and as number and percentage for categorized data. Comparison between two independent groups for nonparametric data was done using Wilcoxon’s rank sum test and χ2-test to study the association between each two variables or comparison between two independent groups regarding the categorized data. The probability of error P value less than or equal to 0.05 was considered statistically significant, and at P value of less than or equal to 0.01 and less than or equal to 0.001 was considered highly significant. P value more than 0.05 was considered statistically nonsignificant. Diagnostic validity test [diagnostic sensitivity and specificity, positive and negative predictive values (PPV and NPV), and test efficacy] was applied. κ-Test was done to measure the agreement between two methods (<0=poor agreement, 0.0–0.20=slight agreement, 0.21–0.40=fair agreement, 0.41–0.60=moderate agreement, 0.61–0.80=substantial agreement, and 0.81–1.00=almost perfect agreement).


  Results Top


The included 50 patients’ demographic, clinical, and laboratory tests are summarized in [Table 1]. Invasive fungal disease was detected in 20 of the 50 (40%) studied patients using the BACTEC Ped Plus blood culture bottles. Candida albicans constituted 75% (15/20) of the isolated fungal pathogens. Aspergillus fumigatus was isolated from 20% (4/20) of positive blood cultures, and Penicillium spp. was isolated from the remaining 5% (1/20) ([Figure 1]).
Table 1 Demographic, clinical, and some laboratory data of all 50 cases

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Figure 1 Results of blood culture-isolated fungi and species identification.

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The RT-PCR assay using the panfungal marker was able to detect IFI in 64% (32/50) of the studied cases. The remaining 36% (18/50) had PCR-negative results. There was a statistically moderate agreement between the results of blood culture and that of real-time PCR regarding the detection of the IFI (κ=0.545) ([Table 2]). The 20 cases that were positive by the blood culture were also positive by the panfungal RT-PCR assay. Yet, 12 (40%) of 30 negative blood culture cases were found to be positive by the panfungal RT-PCR assay, and the remaining 18 (60%) cases were negative by both tests.
Table 2 Agreement between the results of blood culture and real-time polymerase chain reaction

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The diagnostic performance of blood culture was calculated considering RT-PCR for the detection of the panfungal marker as the gold standard. The sensitivity of the routine blood culture was found to be 62.5%, the specificity was 100%, the PPV was 100%, and the NPV was 60%. On the contrary, as we compared the results of PCR for panfungal marker with those of blood culture, the PCR was found to have 100% sensitivity, 60% specificity, PPV of 62.5%, and NPV of 100%.

No significant difference was observed between the PCR-positive and the PCR-negative cases regarding age, the duration of the disease, neutrophil count, the duration of neutropenia, CRP levels, the duration of fever, and the number and duration of antibiotics intake (P>0.05) ([Table 3]).
Table 3 Comparison between polymerase chain reaction-positive and polymerase chain reaction-negative cases regarding the demographic, clinical, and laboratory data

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A highly significant association was observed between the RT-PCR using the panfungal marker and the EORTC/MSG classification (P<0.01) ([Table 4]).
Table 4 Association between European Organization for Research and Treatment of Cancer/Mycoses Study Group classification of patients and polymerase chain reaction results

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  Discussion Top


In the present work, 40% of the studied children with different hematological malignancies had IFIs confirmed by both blood culture and RT-PCR using the panfungal marker. This is much higher than the results obtained by Badiee et al. [1] who were able to diagnose IFI among 10.6% of their studied immunocompromised patients at Nemazi Hospital, southern Iran. Possible reasons for the higher results of positive blood culture in the current study are not clear. A number of factors could be involved, besides known risk factors for IFIs like malignancies, transplantation, and chemotherapy, fungi thrive well in Egypt owing to warm and humid climate. Moreover, people in general are exposed to high conidial burden in the environment. The large economically deprived section, malnutrition, quackery practice, misuse of steroids and antibiotics, late presentation to the clinics, and unprotected construction activities in the hospitals also contribute for this high burden.

C. albicans was found to be the most frequently isolated fungal pathogen (75%) from cases with IFIs followed by A. fumigatus (20%) and Penicillium spp. (5%). This finding is in accordance with the studies of Kovacicova et al. [11], Sogaard et al. [12], and Badiee et al. [13] who found that C. albicans was the most frequently isolated yeast from cases with IFIs accounting for 81, 55, and 72.2%, respectively. However, in the study of Pagano et al. [14], the authors found that molds (especially Aspergillus spp.) are the most common causes of IFI among patients with acute myeloid leukemia.

In the current work, 20 (40%) of the 50 studied patients had IFI diagnosed by blood culture and panfungal RT-PCR: 12 patients (12/50, 24%) were diagnosed only by the panfungal RT-PCR, whereas 18 (36%) patients had negative results by both tests. There was a statistically moderate agreement between the results of blood culture and that of the panfungal RT-PCR regarding the diagnosis of IFI (κ=0.545). Compared with the RT-PCR, the sensitivity of the conventional blood culture was found to be 62.5%, the specificity was 100%, the PPV was 100%, and NPV was 60%. On the contrary, compared with blood culture, the sensitivity of the RT-PCR, using the panfungal marker, was found to be 100%, the specificity was 60%, the PPV was 62.5%, and the NPV was 100%. Similarly, Selim et al. [15] revealed that the sensitivity of blood culture was 62% and the specificity was 100%, and that the sensitivity of panfungal PCR was 100% and the specificity was 40%. On the contrary, El-Sayed et al. [16] found that there was no agreement between the results of blood culture and that of the panfungal PCR assay. Compared with the panfungal PCR, the authors found that the blood culture has a sensitivity of 12%, specificity of 100%, PPV of 100%, and a NPV of 46%.

In our study, there was no significant difference between positive and negative PCR cases regarding the absolute neutrophil count. This is not in accordance with Donowitz et al. [17] who stated that the risk for IFI increases substantially as the absolute neutrophil count declines to less than 500 cells/mm3 and increases further when the count declines to less than 100 cells/mm3. Moreover, Portugal et al. [18] reported that the severity of neutropenia is a good predictor for invasive mold infection in patients with acute myeloid leukemia. The discrepancy between our results and those of the previous studies may be because of the fact that all our patients were extremely neutropenic (median=0.2×103/µl).Unlike the previous findings of Villarroel et al. [19] who reported that fever persisting at day 4 of admission, together with CRP level more than 90 mg/l, significantly increased the risk for IFI in children with cancer, we found insignificant difference between PCR-positive and PCR-negative cases regarding the levels of CRP.

In the present work, we observed no statistically significant difference between the PCR-positive and PCR-negative cases regarding the intake of antibiotics. However, Rowen et al. [20] demonstrated that the intake of antibiotics for more than 14 days increased the risk for fungal infection. Moreover, Saiman et al. [21] added that loss of normal Gastrointestinal tract (GIT) flora owing to treatment with antibiotics may facilitate Candida infection. Our results confirmed the previous findings of Dutta et al. [22] who stated that there is no decline in positive PCR results with the use of antifungal drugs.

A highly significant association was observed in our study between the results of the RT-PCR and the EORTC/MSG classification. All the 20 proven/probable cases were positive by the panfungal PCR assay, and of the 30 possible cases, only 12 turned to be positive by the panfungal PCR assay. Landlinger et al. [8] had concentrated on this correlation between PCR-positive patients and the presence of established criteria of IFI according to the EORTC classification. The authors used the panfungal PCR to investigate 150 febrile neutropenic episodes in pediatric patients with high risk of IFI using. All proven, probable IFI cases and all but one case with possible IFI were PCR-positive.


  Conclusion Top


From the present study, we concluded that the RT-PCR assay for the detection of the panfungal marker is rapid, sensitive, and superior to blood culture technique in the detection of IFI. It can be reliably used for screening immunocompromised patients at risk of IFI. It can especially provide greater confidence in excluding a diagnosis of IFIs when negative results are obtained. Thus, it can help prevent unnecessary toxicity resulting from empirical antifungal treatment in individuals who may not be at risk of imminent fungal disease. However, despite its high sensitivity, the RT-PCR for the detection of the panfungal marker lacks specificity making the interpretation of positive results a challenge.

Acknowledgements

The authors extend their appreciation to the microbiology laboratory specialists at Ain Shams University Hospital for their cooperation and sincere help.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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