Int J Hematol DOI 10.1007/s12185-014-1597-8
CASE REPORT
Serial monitoring of Mucorales DNA load in serum samples of a patient with disseminated mucormycosis after allogeneic bone marrow transplantation Tomonari Shigemura • Yozo Nakazawa • Kazuyuki Matsuda • Kenji Sano • Takashi Yaguchi • Mitsuo Motobayashi • Shoji Saito • Shunsuke Noda • Norimoto Kobayashi • Kazunaga Agematsu • Takayuki Honda • Kenichi Koike
Received: 28 February 2014 / Revised: 30 April 2014 / Accepted: 30 April 2014 Ó The Japanese Society of Hematology 2014
Abstract Mucormycosis is a fatal complication in immunocompromised patients, and is additionally difficult to diagnose due to the lack of useful serum biomarkers. Using a quantitative PCR approach, we retrospectively analyzed Mucorales DNA load in sera collected serially from a 3-year-old patient with chronic granulomatous disease, who died of multi-organ failure probably due to dissemination of Rhizomucor pusillus, which was detected from necropsy specimens. Mucorales DNA load was below the detection limit on days 9, 2, and 4 after unrelated bone marrow transplantation. Rhizomucor DNA was first detected on day 14 (1.6 9 103 copies/ mL), and subsequently fluctuated between 1.3 9 103 and 37.2 9 103 copies/mL until day 43. Rhizomucor achieved a peak value of 940.0 9 103 copies/mL on day 48 the day before death. The detection or fluctuation of Rhizomucor DNA appeared to be associated with corticosteroid dosages or C-reactive protein levels. This specific, noninvasive, and highly quantitative assay may be useful for the
T. Shigemura Y. Nakazawa (&) M. Motobayashi S. Saito S. Noda N. Kobayashi K. Agematsu K. Koike Department of Pediatrics, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan e-mail: [email protected] K. Matsuda K. Sano T. Honda Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan T. Yaguchi Medical Mycology Research Center, Chiba University, Chiba, Japan K. Agematsu Department of Infection and Host Defense, Graduate School of Medicine, Shinshu University, Matsumoto, Japan
early diagnosis of mucormycosis and prediction of disease progression. Keywords Mucormycosis Rhizomucor pusillus Chronic granulomatous disease Serum biomarker Hematopoietic stem cell transplantation
Introduction Mucormycosis is a rapid, aggressive, and angioinvasive infection caused by fungi of the order Mucorales [1, 2]. A meta-analysis showed that 96 % of patients with disseminated disease had died [3]. The high mortality may be due to the lack of serum biomarkers useful for early diagnosis and prediction of disease progression [4, 5]. We present a child with chronic granulomatous disease (CGD) in whom a fatal disseminated mucormycosis developed after allogeneic hematopoietic stem cell transplantation (HSCT). We retrospectively analyzed the Mucorales DNA load in the patient‘s sera collected consecutively using highly quantitative PCR.
Case report A 3-year-old boy with a history of recurrent perianal abscess since the age of 6 months was admitted to our hospital to receive HSCT. At the age of 2 years, he had suffered from severe pneumonia of unknown etiology. His younger sister had suffered from invasive aspergillus pneumonia shortly after birth. They were diagnosed with p67phox-deficient CGD [6]. His sister achieved a marked amelioration of the invasive pulmonary aspergillosis after unrelated cord blood transplantation [7].
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Fig. 1 Clinical course before and after bone marrow transplantation. mPSL methylprednisolone, CRP C-reactive protein, qPCR quantitative PCR, PIPC/TAZ tazobactam/piperacillin, MEPM meropenem, LZD linezolid, TEIC teicoplanin, CFPM cefepime, MCFG micafungin
On admission, ophthalmological, otorhinopharyngeal, and dental examinations revealed no infectious foci. Contrast-enhanced, whole-body CT scan revealed no signs of infection, granuloma, or thrombus. Aspergillus galactomannan antigen and b-D-glucan were undetectable in his serum (cutoff values, \0.5 and \2.84 lg/mL, respectively). Because of the unavailability of human leukocyte antigen (HLA)-identical family donors, bone marrow cells from a genotypically HLA-matched unrelated donor were transplanted after a preparative regimen comprising rabbit anti-thymocyte globulin (ATG, 2.5 mg/kg/day for 2 days), fludarabine (30 mg/m2/day for 6 days), cyclophosphamide (50 mg/kg/day for 2 days), melphalan (80 mg/m2/day for 1 day), and total body irradiation (2 Gy/day for 2 days). Tacrolimus and short-term methotrexate were used for graft-versus-host disease prophylaxis. Micafungin at 5 mg/ kg was infused daily, beginning 8 days before HSCT, for prophylaxis against fungal infection. From day 3 after HSCT, high fever persisted with increased levels of C-reactive protein (CRP, 2.9–7.7 mg/ dL), and on day 10 cough and abdominal pain developed followed by further increase of CRP levels (Fig. 1). On day 13, a skin rash appeared on his palms and soles, and his cough was exacerbated. Methylprednisolone (mPSL) was administered at 12 mg (1.0 mg/kg) per day for suspected engraftment syndrome, and on day 15, the dose was increased to 24 mg/day (Fig. 1). On day 16, abdominal pain was exacerbated, hematuria appeared, and CRP levels exceeded 20 mg/dL (Fig. 1). Enhanced chest CT revealed atelectasis and pulmonary thrombosis in the lower left lobe of the lung and bilateral pleural effusion. A third of the
123
thrombosed area appeared as a wedge-shaped contrastenhanced defect (Fig. 2a). Abdominal CT revealed an enlarged, nonenhancing left kidney (Fig. 2b). Repeated cultures of blood, urine, and stool were negative for fungi and bacteria. Galactomannan antigen and b-D-glucan remained undetectable. D-dimer levels were elevated up to 5.5 lg/mL (normal \1.0 lg/mL) despite intensive anticoagulant therapy. On day 32, he developed hemiparesis and fell unconscious. CT and MRI of the brain revealed bilateral hemorrhagic infarction in the frontal and left parietal lobes (Fig. 2c). On day 36, the mPSL dose was increased up to 20 mg/day because the plasma levels of tacrolimus became unstable on account of a left renal infarction (Fig. 1). On day 49, he died of multiple organ failure with the maximum CRP level (30.2 mg/dL). Histopathological examination of necropsy specimens obtained from both kidneys revealed coagulation necrosis in the entire tissue of the left kidney and fungal hyphae infiltrating the tubulointerstitial area of the right kidney and extending into the interlobular artery. The fungal hyphae showed broad, irregular, aseptate, thin-walled, hyaline hyphae with a very rare angle branching of 90° (Fig. 2d). The fungus was identified as Rhizomucor pusillus by sequence analysis of the internal transcribed spacer region. Materials and methods We performed blood chemistry tests daily between day 9 before and day 49 after HSCT, cryopreserved excess sera at -30 °C until use. DNA was extracted from 200 lL each of the sera.
Quantitative PCR assay for mucormycosis
Fig. 2 Enhanced CT on day 16 after hematopoietic stem cell transplantation shows a a wedge-shaped contrast-enhanced defect in the lower left lobe of lung and b enlarged, absent perfusion in the left kidney. c Brain CT scan on day 32 demonstrates ring-formed bleeding
with perilesional low-density area in the frontal and left parietal lobes. d Photomicrograph of necropsy specimen from kidney shows fungal hyphae with a very rare angle branching of 90° (periodic acid-Schiff stain 9400)
Millon et al. [8] developed a qPCR assay to detect circulating DNA of Mucor/Rhizopus, Lichtheimia, or Rhizomucor. The sequences of primers and probes can be found on the US Environmental Protection Agency website (http:// www.epa.gov/nerlcwww/moldtech.htm#primers) [8]. PCR reactions were run on an ABI 7900HT (Applied Biosystems). The thermal cycling conditions were as follows: an initial denaturation step at 95 °C for 10 min, followed by 50 cycles of 15 s at 95 °C, and 1 min at 60 °C. Although Millon et al. determined the relative amount of Mucorales DNA from the number of PCR cycles in their qPCR assay, we measured the absolute amount to improve quantization in our qPCR assay. Briefly, each assay was designed against a 6-point standard curve constructed using Mucorales-specific sequence-containing plasmids at concentrations ranging from 106 to 10 copies. The threshold cycle value for each sample was plotted on the standard curve, and the copy number of Mucorales DNA was calculated and expressed as the copy number per milliliter of serum. We confirmed
positivity of our qPCR assay for Rhizomucor pusillus, which had been detected from patient’s necropsy specimen, using DNA extracted from one obtained from the National BioResource Project in Japan (http://www.nbrp.jp/). The detection limit of the qPCR assay was 1.0 9 103 copies/mL.
Results We measured the Mucorales DNA load in 9 serum samples collected between day -9 and day 48 after HSCT, as shown in Fig. 1. The DNA load was below the detection limit (\1.0 9 103 copies/mL) on days -9, 2, and 4. A small amount of Rhizomucor DNA was first detected on day 14 (1.6 9 103 copies/mL). The DNA load increased to 19.1 9 103 copies/mL on day 21 through incremental increases in mPSL dose, then gradually decreased to 1.3 9 103 copies/mL by day 36 accompanied by a reduction of mPSL. However, on day 43, the DNA load was
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again elevated to 37.2 9 103 copies/mL 7 days after the increase of mPSL to 30 mg/day. The circulating Rhizomucor showed a peak value of 940.0 9 103 copies/mL on day 48 (a day before the patient’s death). Similarly, the longitudinal change of Rhizomucor DNA load was associated with that of CRP levels to some extent (Fig. 1).
fluctuation of DNA load appeared to be related to that of CRP levels. These results indicate that the quantification of Rhizomucor may predict mucormycosis disease activity. In conclusion, disseminated mucormycosis is a fatal complication of allogeneic HSCT. This modified qPCR assay is specific, noninvasive, and highly quantitative, thus it can potentially be used for the early diagnosis of mucormycosis and prediction of disease progression.
Discussion Conflict of interest We declare that we have no conflicts of interest.
A definitive diagnosis of mucormycosis is based on histopathological findings, molecular detection, and/or a positive culture in the affected tissue. In most cases, however, deep tissue samples cannot be obtained from suspected patients because of HSCT-associated or mucormycosis-induced comorbidities such as thrombocytopenia and coagulopathy. Unlike those for aspergillosis and candidiasis, there are no specific or useful serum biomarkers for diagnosing mucormycosis [4]. Thus, the antemortem diagnosis is difficult to achieve in most cases [1, 2]. Our case was diagnosed by histopathological examination of a necropsy specimen. In this case, elevation of CRP levels was observed almost concomitantly with disease onset and progression (Fig. 1). Ng et al. reported that serial measurements of CRP are valuable in monitoring the treatment for mucormycosis [9]. However, the disease and/or treatment monitoring of mucormycosis by CRP measurements would be inadequate particularly to HSCT due to the nonspecificity. Millon et al. [8] developed a qPCR assay targeting Mucor/Rhizopus, Lichtheimia, and Rhizomucor species, which account for nearly 75 % of patients with mucormycosis post-HSCT [10, 11]. The assay detected Mucorales in the serum of 9 of 10 patients (including one allogeneic HSCT patient) between 68 and 3 days before a definitive diagnosis by histopathological examination and/ or positive culture [8]. In this study, we improved their assay in which Mucorales DNA was expressed as the number of copies per milliliter to improve quantization for universal use. The qPCR detected Rhizomucor DNA in the patient’s serum 7 days earlier, at the onset of the first radiological sign (Fig. 1). Interestingly, the Rhizomucor DNA load in the patient’s sera increased significantly 7 days after the mPSL dose was doubled on day 13 and day 36 (Fig. 1). Rhizomucor DNA load reached the maximal level a day before the patient’s death. Moreover, the
123
References 1. Kontoyiannis DP, Wessel VC, Bodey GP, Rolston KV. Zygomycosis in the 1990s in a tertiary-care cancer center. Clin Infect Dis. 2000;30:851–6. 2. Chamilos G, Lewis RE, Kontoyiannis DP. Delaying amphotericin B-based frontline therapy significantly increases mortality among patients with hematologic malignancy who have zygomycosis. Clin Infect Dis. 2008;47:503–9. 3. Roden MM, Zaoutis TE, Buchanan WL, et al. Epidemiology and outcome of zygomycosis: a review of 929 reported cases. Clin Infect Dis. 2005;41:634–53. 4. Kontoyiannis DP, Lewis RE, Lortholary O, et al. Future directions in mucormycosis research. Clin Infect Dis. 2012;54(Suppl 1): S79–85. 5. Kurosawa M, Yonezumi M, Hashino S, et al. Epidemiology and treatment outcome of invasive fungal infections in patients with hematological malignancies. Int J Hematol. 2012;96:748–57. 6. Wada T, Muraoka M, Toma T, et al. Rapid detection of intracellular p47phox and p67phox by flow cytometry; useful screening tests for chronic granulomatous disease. J Clin Immunol. 2013;33:857–64. 7. Shigemura T, Nakazawa Y, Yoshikawa K, et al. Successful cord blood transplantation after repeated transfusions of unmobilized neutrophils in addition to antifungal treatment in an infant with chronic granulomatous disease complicated by invasive pulmonary aspergillosis. Transfusion. 2013; in press. 8. Millon L, Larosa F, Lepiller Q, et al. Quantitative polymerase chain reaction detection of circulating DNA in serum for early diagnosis of mucormycosis in immunocompromised patients. Clin Infect Dis. 2013;56:e95–101. 9. Ng PC, Dear PR. Phycomycotic abscesses in a preterm infant. Arch Dis Child. 1989;64:862–4. 10. Neofytos D, Horn D, Anaissie E, et al. Epidemiology and outcome of invasive fungal infection in adult hematopoietic stem cell transplant recipients: analysis of Multicenter Prospective Antifungal Therapy (PATH) Alliance registry. Clin Infect Dis. 2009;48:265–73. 11. Xhaard A, Lanternier F, Porcher R, et al. Mucormycosis after allogeneic haematopoietic stem cell transplantation: a French Multicentre Cohort Study (2003–2008). Clin Microbiol Infect. 2012;18:e396–400.
CASE REPORT
Serial monitoring of Mucorales DNA load in serum samples of a patient with disseminated mucormycosis after allogeneic bone marrow transplantation Tomonari Shigemura • Yozo Nakazawa • Kazuyuki Matsuda • Kenji Sano • Takashi Yaguchi • Mitsuo Motobayashi • Shoji Saito • Shunsuke Noda • Norimoto Kobayashi • Kazunaga Agematsu • Takayuki Honda • Kenichi Koike
Received: 28 February 2014 / Revised: 30 April 2014 / Accepted: 30 April 2014 Ó The Japanese Society of Hematology 2014
Abstract Mucormycosis is a fatal complication in immunocompromised patients, and is additionally difficult to diagnose due to the lack of useful serum biomarkers. Using a quantitative PCR approach, we retrospectively analyzed Mucorales DNA load in sera collected serially from a 3-year-old patient with chronic granulomatous disease, who died of multi-organ failure probably due to dissemination of Rhizomucor pusillus, which was detected from necropsy specimens. Mucorales DNA load was below the detection limit on days 9, 2, and 4 after unrelated bone marrow transplantation. Rhizomucor DNA was first detected on day 14 (1.6 9 103 copies/ mL), and subsequently fluctuated between 1.3 9 103 and 37.2 9 103 copies/mL until day 43. Rhizomucor achieved a peak value of 940.0 9 103 copies/mL on day 48 the day before death. The detection or fluctuation of Rhizomucor DNA appeared to be associated with corticosteroid dosages or C-reactive protein levels. This specific, noninvasive, and highly quantitative assay may be useful for the
T. Shigemura Y. Nakazawa (&) M. Motobayashi S. Saito S. Noda N. Kobayashi K. Agematsu K. Koike Department of Pediatrics, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan e-mail: [email protected] K. Matsuda K. Sano T. Honda Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan T. Yaguchi Medical Mycology Research Center, Chiba University, Chiba, Japan K. Agematsu Department of Infection and Host Defense, Graduate School of Medicine, Shinshu University, Matsumoto, Japan
early diagnosis of mucormycosis and prediction of disease progression. Keywords Mucormycosis Rhizomucor pusillus Chronic granulomatous disease Serum biomarker Hematopoietic stem cell transplantation
Introduction Mucormycosis is a rapid, aggressive, and angioinvasive infection caused by fungi of the order Mucorales [1, 2]. A meta-analysis showed that 96 % of patients with disseminated disease had died [3]. The high mortality may be due to the lack of serum biomarkers useful for early diagnosis and prediction of disease progression [4, 5]. We present a child with chronic granulomatous disease (CGD) in whom a fatal disseminated mucormycosis developed after allogeneic hematopoietic stem cell transplantation (HSCT). We retrospectively analyzed the Mucorales DNA load in the patient‘s sera collected consecutively using highly quantitative PCR.
Case report A 3-year-old boy with a history of recurrent perianal abscess since the age of 6 months was admitted to our hospital to receive HSCT. At the age of 2 years, he had suffered from severe pneumonia of unknown etiology. His younger sister had suffered from invasive aspergillus pneumonia shortly after birth. They were diagnosed with p67phox-deficient CGD [6]. His sister achieved a marked amelioration of the invasive pulmonary aspergillosis after unrelated cord blood transplantation [7].
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T. Shigemura et al.
Fig. 1 Clinical course before and after bone marrow transplantation. mPSL methylprednisolone, CRP C-reactive protein, qPCR quantitative PCR, PIPC/TAZ tazobactam/piperacillin, MEPM meropenem, LZD linezolid, TEIC teicoplanin, CFPM cefepime, MCFG micafungin
On admission, ophthalmological, otorhinopharyngeal, and dental examinations revealed no infectious foci. Contrast-enhanced, whole-body CT scan revealed no signs of infection, granuloma, or thrombus. Aspergillus galactomannan antigen and b-D-glucan were undetectable in his serum (cutoff values, \0.5 and \2.84 lg/mL, respectively). Because of the unavailability of human leukocyte antigen (HLA)-identical family donors, bone marrow cells from a genotypically HLA-matched unrelated donor were transplanted after a preparative regimen comprising rabbit anti-thymocyte globulin (ATG, 2.5 mg/kg/day for 2 days), fludarabine (30 mg/m2/day for 6 days), cyclophosphamide (50 mg/kg/day for 2 days), melphalan (80 mg/m2/day for 1 day), and total body irradiation (2 Gy/day for 2 days). Tacrolimus and short-term methotrexate were used for graft-versus-host disease prophylaxis. Micafungin at 5 mg/ kg was infused daily, beginning 8 days before HSCT, for prophylaxis against fungal infection. From day 3 after HSCT, high fever persisted with increased levels of C-reactive protein (CRP, 2.9–7.7 mg/ dL), and on day 10 cough and abdominal pain developed followed by further increase of CRP levels (Fig. 1). On day 13, a skin rash appeared on his palms and soles, and his cough was exacerbated. Methylprednisolone (mPSL) was administered at 12 mg (1.0 mg/kg) per day for suspected engraftment syndrome, and on day 15, the dose was increased to 24 mg/day (Fig. 1). On day 16, abdominal pain was exacerbated, hematuria appeared, and CRP levels exceeded 20 mg/dL (Fig. 1). Enhanced chest CT revealed atelectasis and pulmonary thrombosis in the lower left lobe of the lung and bilateral pleural effusion. A third of the
123
thrombosed area appeared as a wedge-shaped contrastenhanced defect (Fig. 2a). Abdominal CT revealed an enlarged, nonenhancing left kidney (Fig. 2b). Repeated cultures of blood, urine, and stool were negative for fungi and bacteria. Galactomannan antigen and b-D-glucan remained undetectable. D-dimer levels were elevated up to 5.5 lg/mL (normal \1.0 lg/mL) despite intensive anticoagulant therapy. On day 32, he developed hemiparesis and fell unconscious. CT and MRI of the brain revealed bilateral hemorrhagic infarction in the frontal and left parietal lobes (Fig. 2c). On day 36, the mPSL dose was increased up to 20 mg/day because the plasma levels of tacrolimus became unstable on account of a left renal infarction (Fig. 1). On day 49, he died of multiple organ failure with the maximum CRP level (30.2 mg/dL). Histopathological examination of necropsy specimens obtained from both kidneys revealed coagulation necrosis in the entire tissue of the left kidney and fungal hyphae infiltrating the tubulointerstitial area of the right kidney and extending into the interlobular artery. The fungal hyphae showed broad, irregular, aseptate, thin-walled, hyaline hyphae with a very rare angle branching of 90° (Fig. 2d). The fungus was identified as Rhizomucor pusillus by sequence analysis of the internal transcribed spacer region. Materials and methods We performed blood chemistry tests daily between day 9 before and day 49 after HSCT, cryopreserved excess sera at -30 °C until use. DNA was extracted from 200 lL each of the sera.
Quantitative PCR assay for mucormycosis
Fig. 2 Enhanced CT on day 16 after hematopoietic stem cell transplantation shows a a wedge-shaped contrast-enhanced defect in the lower left lobe of lung and b enlarged, absent perfusion in the left kidney. c Brain CT scan on day 32 demonstrates ring-formed bleeding
with perilesional low-density area in the frontal and left parietal lobes. d Photomicrograph of necropsy specimen from kidney shows fungal hyphae with a very rare angle branching of 90° (periodic acid-Schiff stain 9400)
Millon et al. [8] developed a qPCR assay to detect circulating DNA of Mucor/Rhizopus, Lichtheimia, or Rhizomucor. The sequences of primers and probes can be found on the US Environmental Protection Agency website (http:// www.epa.gov/nerlcwww/moldtech.htm#primers) [8]. PCR reactions were run on an ABI 7900HT (Applied Biosystems). The thermal cycling conditions were as follows: an initial denaturation step at 95 °C for 10 min, followed by 50 cycles of 15 s at 95 °C, and 1 min at 60 °C. Although Millon et al. determined the relative amount of Mucorales DNA from the number of PCR cycles in their qPCR assay, we measured the absolute amount to improve quantization in our qPCR assay. Briefly, each assay was designed against a 6-point standard curve constructed using Mucorales-specific sequence-containing plasmids at concentrations ranging from 106 to 10 copies. The threshold cycle value for each sample was plotted on the standard curve, and the copy number of Mucorales DNA was calculated and expressed as the copy number per milliliter of serum. We confirmed
positivity of our qPCR assay for Rhizomucor pusillus, which had been detected from patient’s necropsy specimen, using DNA extracted from one obtained from the National BioResource Project in Japan (http://www.nbrp.jp/). The detection limit of the qPCR assay was 1.0 9 103 copies/mL.
Results We measured the Mucorales DNA load in 9 serum samples collected between day -9 and day 48 after HSCT, as shown in Fig. 1. The DNA load was below the detection limit (\1.0 9 103 copies/mL) on days -9, 2, and 4. A small amount of Rhizomucor DNA was first detected on day 14 (1.6 9 103 copies/mL). The DNA load increased to 19.1 9 103 copies/mL on day 21 through incremental increases in mPSL dose, then gradually decreased to 1.3 9 103 copies/mL by day 36 accompanied by a reduction of mPSL. However, on day 43, the DNA load was
123
T. Shigemura et al.
again elevated to 37.2 9 103 copies/mL 7 days after the increase of mPSL to 30 mg/day. The circulating Rhizomucor showed a peak value of 940.0 9 103 copies/mL on day 48 (a day before the patient’s death). Similarly, the longitudinal change of Rhizomucor DNA load was associated with that of CRP levels to some extent (Fig. 1).
fluctuation of DNA load appeared to be related to that of CRP levels. These results indicate that the quantification of Rhizomucor may predict mucormycosis disease activity. In conclusion, disseminated mucormycosis is a fatal complication of allogeneic HSCT. This modified qPCR assay is specific, noninvasive, and highly quantitative, thus it can potentially be used for the early diagnosis of mucormycosis and prediction of disease progression.
Discussion Conflict of interest We declare that we have no conflicts of interest.
A definitive diagnosis of mucormycosis is based on histopathological findings, molecular detection, and/or a positive culture in the affected tissue. In most cases, however, deep tissue samples cannot be obtained from suspected patients because of HSCT-associated or mucormycosis-induced comorbidities such as thrombocytopenia and coagulopathy. Unlike those for aspergillosis and candidiasis, there are no specific or useful serum biomarkers for diagnosing mucormycosis [4]. Thus, the antemortem diagnosis is difficult to achieve in most cases [1, 2]. Our case was diagnosed by histopathological examination of a necropsy specimen. In this case, elevation of CRP levels was observed almost concomitantly with disease onset and progression (Fig. 1). Ng et al. reported that serial measurements of CRP are valuable in monitoring the treatment for mucormycosis [9]. However, the disease and/or treatment monitoring of mucormycosis by CRP measurements would be inadequate particularly to HSCT due to the nonspecificity. Millon et al. [8] developed a qPCR assay targeting Mucor/Rhizopus, Lichtheimia, and Rhizomucor species, which account for nearly 75 % of patients with mucormycosis post-HSCT [10, 11]. The assay detected Mucorales in the serum of 9 of 10 patients (including one allogeneic HSCT patient) between 68 and 3 days before a definitive diagnosis by histopathological examination and/ or positive culture [8]. In this study, we improved their assay in which Mucorales DNA was expressed as the number of copies per milliliter to improve quantization for universal use. The qPCR detected Rhizomucor DNA in the patient’s serum 7 days earlier, at the onset of the first radiological sign (Fig. 1). Interestingly, the Rhizomucor DNA load in the patient’s sera increased significantly 7 days after the mPSL dose was doubled on day 13 and day 36 (Fig. 1). Rhizomucor DNA load reached the maximal level a day before the patient’s death. Moreover, the
123
References 1. Kontoyiannis DP, Wessel VC, Bodey GP, Rolston KV. Zygomycosis in the 1990s in a tertiary-care cancer center. Clin Infect Dis. 2000;30:851–6. 2. Chamilos G, Lewis RE, Kontoyiannis DP. Delaying amphotericin B-based frontline therapy significantly increases mortality among patients with hematologic malignancy who have zygomycosis. Clin Infect Dis. 2008;47:503–9. 3. Roden MM, Zaoutis TE, Buchanan WL, et al. Epidemiology and outcome of zygomycosis: a review of 929 reported cases. Clin Infect Dis. 2005;41:634–53. 4. Kontoyiannis DP, Lewis RE, Lortholary O, et al. Future directions in mucormycosis research. Clin Infect Dis. 2012;54(Suppl 1): S79–85. 5. Kurosawa M, Yonezumi M, Hashino S, et al. Epidemiology and treatment outcome of invasive fungal infections in patients with hematological malignancies. Int J Hematol. 2012;96:748–57. 6. Wada T, Muraoka M, Toma T, et al. Rapid detection of intracellular p47phox and p67phox by flow cytometry; useful screening tests for chronic granulomatous disease. J Clin Immunol. 2013;33:857–64. 7. Shigemura T, Nakazawa Y, Yoshikawa K, et al. Successful cord blood transplantation after repeated transfusions of unmobilized neutrophils in addition to antifungal treatment in an infant with chronic granulomatous disease complicated by invasive pulmonary aspergillosis. Transfusion. 2013; in press. 8. Millon L, Larosa F, Lepiller Q, et al. Quantitative polymerase chain reaction detection of circulating DNA in serum for early diagnosis of mucormycosis in immunocompromised patients. Clin Infect Dis. 2013;56:e95–101. 9. Ng PC, Dear PR. Phycomycotic abscesses in a preterm infant. Arch Dis Child. 1989;64:862–4. 10. Neofytos D, Horn D, Anaissie E, et al. Epidemiology and outcome of invasive fungal infection in adult hematopoietic stem cell transplant recipients: analysis of Multicenter Prospective Antifungal Therapy (PATH) Alliance registry. Clin Infect Dis. 2009;48:265–73. 11. Xhaard A, Lanternier F, Porcher R, et al. Mucormycosis after allogeneic haematopoietic stem cell transplantation: a French Multicentre Cohort Study (2003–2008). Clin Microbiol Infect. 2012;18:e396–400.
