Detection of imported histoplasmosis in serum of HIV-infected patients using a real-time PCR-based assay

  • M. J. Buitrago
  • J. Berenguer
  • E. Mellado
  • J. L. Rodríguez-Tudela
  • M. Cuenca-Estrella
Concise Article

Abstract

A new real-time PCR-based assay was used for detecting DNA of Histoplasma capsulatum in serum samples collected from four HIV-infected patients with proven histoplasmosis. The assay targeted the ITS1 region of rDNA and its in vitro sensitivity, specificity and reproducibility were evaluated. The technique detected DNA of H. capsulatum in all of the HIV-infected patients with proven histoplasmosis (4/4, 100%). The PCR result was positive for seven of the ten (70%) samples studied. The assay’s specificity was determined to be 100%, since the method was negative for 25 other serum samples (10 from patients with proven aspergillosis and 15 from healthy controls). The PCR assay is a new and promising diagnostic alternative and further investigation is warranted.

Introduction

The incidence of histoplasmosis in Spain has increased in recent years, primarily due to migration of humans from endemic regions and secondarily due to travelers returning from these regions. In Spain, disseminated histoplasmosis is usually diagnosed in HIV-infected patients from endemic areas [1, 2]. A variety of diagnostic methods have been described to date; however, all of these have limitations. Isolating the organism from culture is considered the gold-standard method, but growth of Histoplasma capsulatum is very slow (3–4 weeks) [3], and serologic tests are negative in up to 50% of immunosuppressed patients, especially those with AIDS. Antigen detection in urine and/or serum is a very useful method in the case of patients with disseminated histoplasmosis [4, 5]; however, this test is not accessible in the majority of non-endemic areas.

Conventional PCR assays to detect H. capsulatum DNA have been described previously [6]. Ribosomal DNA has often been used for diagnostic PCR because multiple gene copies improve the assay’s sensitivity. PCR targeted to single-copy genes has also been developed, such as those coding the M antigen [7] and the 100 kDa-like protein [8]. Conventional PCR assay using these methods has been utilized to diagnose disseminated histoplasmosis in an AIDS patient, but while it detected H. capsulatum DNA in respiratory samples it detected none in blood [9].

Quantitative PCR assays, such as real-time PCR, have been proposed for the diagnosis of invasive fungal disease because of their greater sensitivity and specificity compared to conventional PCR, and because the time to diagnosis is shorter than that required by cultures [10]. Here, we report a new specific real-time PCR for the detection of H. capsulatum in HIV-infected patients.

Materials and methods

A clinical strain of H. capsulatum belonging to the microorganism collection of the Spanish National Center of Microbiology (CNM-CM-2721) was included as a quality control strain in each set of experiments. It was used to test the sensitivity and reproducibility of the real-time PCR assay.

The specificity of the technique was also assessed with other fungal species included in the study using previously described methods [11]. The strains analyzed were as follows: one clinical isolate of Coccidioides immitis (CNM-CM-2911), one of Paracoccidioides brasiliensis (CNM-CM-2908), two Blastomyces dermatitidis (ATCC26199, ATCC56220), one Aspergillus fumigatus (CNM-CM-237), one Aspergillus flavus (CNM-CM-2669), one Aspergillus terreus (CNM-CM-2013), one Fusarium verticillioides (CNM-CM-2975), one Fusarium oxysporum (CNM-CM-2914), one Scedosporium prolificans (CNM-CM-2387), one Scedosporium apiospermum (CNM-CM-2530), and one Candida albicans (ATCC64551). In addition, human and murine genomic DNA (Promega, Madrid, Spain) was included in the experiments. The specificity of primers and probes was also assessed by analyzing and comparing the nucleotide sequence of the ITS regions of 1,800 isolates of 150 distinct fungal species. These sequences belong to the database of the Department of Mycology of the Spanish National Center for Microbiology, and the analysis was performed with the help of Fingerprinting II Informatix software, version 3.0 (BioRad, Madrid, Spain).

DNA extraction from H. capsulatum, C. immitis, P. brasiliensis and B. dermatitidis mycelia was performed in biosafety level III facilities, in compliance with Spanish laws (Real Decreto 664/1997) and following routine methods [12]. DNA extraction from the other filamentous and yeast species was performed following the method described previously by Tang et al. [12].

Primers and probes were designed on the basis of the nucleotide sequence of the ITS1 rDNA region for 20 strains of H. capsulatum. Primers and probes selected were subjected to a BLAST search in the GenBank sequence database (http://www.ncbi.nih.gov/Genbank/) to avoid cross-homology with other microorganisms. LightCycler Probe Design software, version 1.0 (Roche Applied Science, Madrid, Spain) was used to find the best primers and probes to perform the real-time PCR assay. The forward primer selected was 5′-CCACCCTTGTCTACC-3′ (primer Hcits1-1) and the reverse primer was 5′-GGAACCAAGAGATCCGT-3′ (primer Hcits1-2). The amplified fragment was 182 bp in length. The probes were marked using fluoresce resonance energy transfer (FRET) technology with HC1-Fluos 5′GTCGGTGAACGATTGGCGT3′-LCFluorescein and HC1-Red 5′LCRED640-GAGCATGAGAGCGATAATAATCCAGT3′-(Tibmolbiol) (Roche).

PCR reactions were performed in the LightCycler 2.0 System (Roche). The kit LightCycler Fast Start DNA Master Hybridization Probes (Roche) was used as described by the manufacturer. PCR products were subjected to electrophoresis in 2% agarose gels (Pronadisa, Madrid, Spain), following the protocols of Sambrook et al. [13], in order to verify the PCR results. Amplified fragments were sequenced using the ABI Prism 377 DNA sequencer (Applied Biosystem, Madrid, Spain), and the sequences obtained were compared to the H. capsulatum ITS1 sequence in the laboratory database. Each PCR run contained negative controls and positive controls in the form of genomic DNA from H. capsulatum CNM-CM-2721 at distinct concentrations.

A standard curve was constructed using the PCR results of eight repetitions of different dilutions of genomic DNA of H. capsulatum CNM-CM-2721. Dilutions ranged between 10 ng and 1 fg DNA/μl. The crossing-point values (i.e., the cycle in which the fluorescence is detected) were plotted against the logarithmically converted DNA concentrations, and the linear regression coefficient was calculated. The reproducibility of the assay was determined by calculating the average value and 99% confidence interval of the crossing point for each DNA concentration. The reproducibility was defined as a percentage of values inside the confidence interval. In addition, a range of assay validation was calculated per DNA concentration taking the average values and confidence intervals into account.

Sera from four HIV-positive patients with proven histoplasmosis from endemic regions were collected at the Hospital Gregorio Marañón, a 1,750-bed tertiary referral general teaching institution in Madrid. Samples were stored at −80°C prior to being used. Proven histoplasmosis was defined as the isolation of fungi in culture. Table 1 shows the clinical characteristics of the patients. In addition, 25 serum samples were tested for the specificity study (10 from aspergillosis patients and 15 from healthy controls).
Table 1

Demographics, clinical manifestations, complementary examinations, therapy and outcome of patients with AIDS-related histoplasmosis

Patient characteristics/identification

Patient 1

Patient 2

Patient 3

Patient 4

Age in years

30

45

32

44

Sex

Male

Female

Male

Male

Country of origin

Ecuador

Ecuador

Venezuela

Ecuador

HIV transmission category

Heterosexual

Heterosexual

Homosexual

Unknown

Date of HIV diagnosis

22 June 2001

29 September 2003

31 January 2004

29 August 2003

Date of histoplasmosis diagnosis

22 June 2001

24 October 2003

31 January 2004

24 April 2004

Clinical manifestations

Fever, malaise, weight loss

Isolated laterocervical adenopathy

Fever, malaise, weight loss

Fever, malaise, headache

Baseline CD4+ cell counta

112/mm3

421/mm3

4/mm3

49/mm3

Sites of culture of H. capsulatum

Blood, liver biopsy

Lymph node

Blood, bone marrow, skin

Blood, CSF, bone marrow, lymph node

Diagnosis

Disseminated histoplasmosis

Focal histoplasmosisb

Disseminated histoplasmosis

Disseminated histoplasmosis

Antifungal therapy

Liposomal Amphotericin B and oral itraconazole

Oral itraconazole

Liposomal amphotericin B

Liposomal amphotericin B

Initiation of therapy

26 June 2001

27 November 2003

3 February 2004

17 October 2003

Discontinuation of therapy

18 July 2003

22 September 2004

Still on medication

Lost to FU (travelled to Ecuador)

Last FU visit

10 March 2005

22 February 2005

16 March 2005

20 July 2004

Status

Well and alive

Well and alive

Well and alive

Lost to FU (travelled to Ecuador)

Last visit CD4+ cell count

378/mm3

500/mm3

140/mm3

160/mm3

aAt the time of histoplasmosis diagnosis

bBlood cultures were invariably negative and fever, malaise and weight loss were not observed.

CSF cerebrospinal fluid, FU follow-up

DNA was extracted from sera using the NucleoSpinBlood Kit (Macherey-Nalgen, Cultek, Madrid, Spain). Amounts of fungal DNA in the clinical samples were calculated and validated based on the standard curve described above. Amplification products were sequenced and compared with those in the DNA database, as stated above. Experiments were repeated three times on three separate days.

In addition, precipitating antibodies against H. capsulatum was detected in patient sera using an immunodiffusion test according to the manufacturer’s recommendations (ID Fungal Antibody System, Immuno-Mycologics, Norman USA).

Results and discussion

The real-time PCR we designed was able to specifically detect H. capsulatum DNA in vitro. The assay sensitivity was 5 fg of DNA per μl of sample. The determination coefficient (r2) of the linear regression between the crossing point values and different dilutions of genomic DNA of H. capsulatum CNM-CM-2721 was −0.99 (p < 0.01). The reproducibility was 100% for the six highest DNA concentrations. However, the reproducibility decreased to 62.5% for the DNA concentration of 10 fg. Below 5 fg/μl, the regression lost its linearity. The average amplification efficiency was 1.8. No positive signal was detected when human and mouse DNA and DNA from other fungal species was tested.

A total of 35 clinical serum samples were analyzed: ten from patients with proven histoplasmosis, ten from patients with aspergillosis, and 15 from healthy controls. The assay’s specificity was 100%, since no amplification was detected in 25 patients without histoplasmosis (10 aspergillosis and 15 healthy controls). Sensitivity was evaluated using the ten serum samples from four patients with proven histoplasmosis. The PCR result was positive for seven of the ten (70%) samples studied. The PCR assay detected DNA of H. capsulatum in the samples of all patients with proven histoplasmosis (4/4, 100%). The average DNA concentration per μl of serum analyzed was 28.49 fg. The serological test was positive for one of four (25%) patients.

Samples from patient 1 and patient 4 were invariably positive by PCR. They were taken at the same time or not long after histoplasmosis was diagnosed. Immunodiffusion results were negative for both patients. Two samples collected from patient 3 a short time following the histoplasmosis diagnosis were analyzed, but no more than one was positive by PCR amplification. Patient 3 was the only one who exhibited detectable antibodies by immunodiffusion (M band) against H. capsulatum. Patient 2 was diagnosed as having focal histoplasmosis with a laterocervical adenopathy, and two of four serum samples investigated were positive by the PCR assay. Immunodiffusion results were negative in patient 2.

The diagnosis of disseminated histoplasmosis requires a high index of clinical suspicion [14], especially in non-endemic regions. A delay in diagnosis may lead to a fatal outcome in more severe cases, but rapid diagnosis is hampered by the slow growth of the fungus and the need for trained mycologists capable of confirming visual identification. Serologic tests also have their limitations [3]. PCR-based detection methods have been described for H. capsulatum, the majority of which used conventional PCR targeted to multicopy or single copy genes [6, 7, 8, 10, 15]. Sensitivity varied among the different assays described, and little data on testing clinical samples has been reported. One real-time PCR assay has been described that targeted the ITS1 region of rDNA [10]. This assay was designed to identify H. capsulatum from cultures. The researchers identified 34 H. capsulatum isolates and checked the sensitivity of the assay using clinical samples of three patients suffering from proven histoplasmosis. DNA of H. capsulatum was detected in respiratory tract specimens, bone marrow biopsies and blood, but serum samples were not included in the study. No data regarding the reproducibility and sensitivity of the PCR assay were reported [10].

We have designed a rapid method for detecting H. capsulatum DNA based on real-time PCR, which produces results in just a few hours. Specific FRET probes targeting the ITS1 regions of rDNA were designed. These regions are multicopy and species-specific, which increases the sensitivity of the assay and solves the problem of contamination or lack of specificity. The PCR assay described in this work was specific for detecting H. capsulatum DNA, and it detected up to 5 fg of fungal DNA per μl of sample. We detected fungal DNA in 70% of serum samples from patients with proven histoplasmosis and all patients had at least one positive result. Serology was positive in one patient only, thus confirming the low usefulness of antibody detection for the diagnosis of histoplasmosis in HIV patients. The assay had a specificity of 100%, since it was invariably negative with the sera of healthy controls and patients with aspergillosis.

These findings require further investigation. New studies should be carried out including a higher number of patients and other clinical samples such as respiratory tract specimens and blood.

Notes

Acknowledgements

This work was supported in part by research projects 1021/04, from the Instituto de Salud Carlos III, and 1397/04, from Fundación Ramón Areces, Madrid, Spain.

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Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • M. J. Buitrago
    • 1
  • J. Berenguer
    • 2
  • E. Mellado
    • 1
  • J. L. Rodríguez-Tudela
    • 1
  • M. Cuenca-Estrella
    • 1
  1. 1.Servicio de Micología, Centro Nacional de MicrobiologíaInstituto de Salud Carlos IIIMadridSpain
  2. 2.Unidad de Enfermedades InfecciosasHospital General Gregorio MarañonMadridSpain

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