Five laboratories were involved in this study. Four of them are located in endemic countries (Argentina, Brazil, Colombia, and México) and one in a non-endemic region (Spain); all are members of the MICOMOL-CYTED network. The centers have both clinical and scientific expertise in the field and use PCR techniques in routine laboratory tasks.15,20 They have been designated with the following numerical code: (Lab. 1) Unidad de Micología Médica y Experimental, Corporación para Investigaciones Biológicas, Medellín, Colombia; (Lab. 2) Departamento de Microbiología y Parasitología, UNAM, México D.F., Mexico; (Lab. 3) Laboratório de Micologia, Instituto de Pesquisa Clinica Evandro Chagas, Fundação Osvaldo Cruz, Rio de Janeiro, Brazil; (Lab. 4) Servicio Micosis Profundas, Departamento de Micología, INEI-ANLIS Dr. Carlos G. Malbrán, Buenos Aires, Argentina; and (Lab. 5) Servicio de Micología, Instituto de Salud Carlos III, Madrid, Spain.

Two of these laboratories (Lab. 2 and Lab. 4) contributed with two different protocols and were designated as Lab. 2(1), Lab. 2(2), and Lab. 4(1), Lab. 4(2), respectively. The rest of the centers contributed with a single protocol.

A blind panel with ten tubes containing different concentrations of fungal DNA was sent to each participant laboratory in May 2011 (see Table 1). Tubes with different concentrations of H. capsulatum DNA were included together with tubes containing water as negative controls and a tube with Paracoccidioides brasiliensis DNA. To avoid DNA degradation in freeze–thaw, aliquots containing samples were frozen and sent to the laboratories. Each laboratory thawed the panel samples only to perform the assays. To validate the panels, initial testing was performed by the organizing group before their distribution. The receiving centers were asked to confirm the panel reception, comment on its state and keep the tubes frozen at −20°C until tested.

Clinical strains of H. capsulatum (CNM-CM-2721) and P. brasiliensis (CNM-CM-2908) belonging to the microorganism collection of the Spanish National Centre of Microbiology were used to extract DNA.

Each participant center used their own strains to include as positive controls in their respective PCR assays.

DNA extraction from the strains was performed in biosafety level III facilities in compliance with Spanish law.1 Briefly, the strains were grown statically at 30°C in 10ml of GYEP broth (2% glucose, 0.3% yeast extract, 1% peptone) for a week. Mycelia were harvested, dried and introduced in a Falcon tube with six glass beads of 4mm diameter (Sigma–Aldrich Química, Madrid, Spain). The mycelia were subjected to two cycles of freezing in liquid nitrogen for 30s and vortex for 30s to obtain a fine powder. Then, the kit “DNeasy Plant Mini Kit” (Qiagen, Izasa, Madrid, Spain) was employed to extract DNA from the obtained powder following the manufacturer's instructions. The quality and amount of DNA obtained was checked spectrophotometrically.

The participants were told to use their current amplification systems. A total of seven protocols were tested. These protocols were based on conventional, nested and real time PCR and targeted different DNA regions. The enzymes, the target and the equipment used by each laboratory are described in Table 2. Reactions were performed in a final volume of 20μl, and 2μl of DNA from each tube in the panel were added to the reaction, with the final DNA concentration in the PCR tube being tenfold lower than in the original tube. Each laboratory included positive and negative controls.

Lab. 2 tested the SCAR220 marker (Sequence-Characterized Amplified Region) specific for H. capsulatum. In this protocol, a 220bp fragment was amplified9 using the primers 1281-1283220F (5′-CATTGTTGGAGGAACCTGCT-3′) and 1281-1283220R (5′-GAGCTGCAGGATGTTTGTTG-3′) in the following conditions: denaturation at 94°C for 3min and then 30 cycles of 94°C for 30s, 55°C for 30s; 72°C for 2min and a final extension step at 72°C for 5min. PCR products were subjected to electrophoresis in agarose gels following the protocols of Sambrook and Russel19 to confirm the PCR results. The amplicons were purified using the QIAQuick purification Kit (Qiagen, Valencia, CA) and sequencing was performed using ABI equipment (Applied Biosystems Inc., Foster City, CA).

Labs. 1, 2, 3 and 4 used the method described by Bialek et al.3 that targeted the 100-kDa-like protein unique in H. capsulatum. This protein has been described as essential for the survival of the pathogen.16 This protocol was a nested conventional PCR assay in which the first round amplified a 391bp fragment and the second round a 210bp fragment. Primers and PCR conditions were as described by Bialek et al.3 PCR results were verified by using electrophoresis in agarose gels.19

Lab. 4 used a real-time PCR assay that targeted the multicopy ITS1 region of the ribosomal DNA and used the specific primers HcITS-54F (5′-ACCCTTGTCTACCGGACCTGTT-3′ and HcITS-204R (5′-TTTTGACTGGATTATTATCGCTCTCA-3′) and the Taqman probe HcITS-155 (5′ FAM-CGGTGAACGATTGGCGTCTGAGC-TAMRA 3′). These primers and probes were designed using the Program Primer Express® Software version 3.0 (Applied Biosystems). Each reaction mixture contained 0.5μM of each primer and 0.2μM of probe. The cycling conditions included the next steps: 2min at 50°C, 10min at 95°C and then 50 cycles as follows: 15s at 95°C and 1min at 60°C. Lab. 5 used a previously described real-time PCR assay4,5 that had been validated in clinical samples of patients with imported histoplamosis.5 This protocol used specific FRET probes that targeted the ITS1 region of ribosomal DNA and included an internal control. For both assays, standard curves were constructed with PCR results from five repetitions of different dilutions of genomic DNA of H. capsulatum. Dilutions ranged between 107 and 1fg DNA/μl. A line (y=mx+b) was constructed by plotting the standard curve of log quantity versus its corresponding CT value, a cycle in which fluorescence becomes detectable from the background. An average value and a 99% confidence interval for crossing-point values obtained for each DNA concentration were calculated.

The protocol based on SCAR marker [Lab. 2(1)] presented the lowest sensitivity for the panel sent (43%) and was not able to detect DNA concentrations of less than 104fg/μl. However, this protocol did not produce false positives or cross-reactions.

The limit of detection was different for the four laboratories that performed this protocol. In Lab. 3 and Lab. 4 the protocols based on the 100-kDa-like protein were able to detect the different H. capsulatum DNA concentrations used, reaching the lowest concentration (10fg/μl). In Lab. 1 and Lab. 2(2), fungal DNA concentrations in the PCR reaction tube of less than 102 and 103fg/μl, respectively, were not detected. The overall sensitivity for the panel using this protocol was 89.5%. The detection limit was established between 103 and 10fg/μl. False positives were not detected in any case.

Both protocols based on real-time PCR were able to detect the lowest concentrations of DNA (10fg/μl).

When DNA concentration was estimated based on the calibration curves, the average of relative errors was 77% for Lab. 4 and 18.5% for Lab. 5 (Table 3).

Neither false positives nor cross-reactions were detected with these protocols.