ABSTRACT PCR has been used as an aid in the diagnosis of invasive aspergillosis for almost 2 decades. A lack of standardization has limited both its acceptance as a diagnostic tool and multicenter clinical evaluations, preventing its inclusion in disease-defining criteria. In 2006, the European Aspergillus PCR Initiative was formed. The aim of the initiative was to provide optimal standardized protocols for the widespread clinical evaluation of the Aspergillus PCR to determine its diagnostic role and allow inclusion in disease diagnosis criteria. Quality control panels were developed and circulated to centers for evaluation of the existing methodology before recommendations based on the initial results were proposed for further panels. The centers were anonymously classified as “compliant” or “noncompliant,” according to whether they had followed the proposed recommendations before the performance parameters were determined and meta-regression analysis was performed. Most PCR amplification systems provided similar detection thresholds, although positivity was a function of the fungal burden. When PCR amplification was combined with DNA extraction, 50% of the centers failed to achieve the same level of detection. Meta-regression analysis showed positive correlations between sensitivity and extraction protocols incorporating the proposed recommendations and the use of bead beating, white cell lysis buffer, and an internal control PCR. The use of elution volumes above 100 μl showed a negative correlation with sensitivity. The efficiency of the Aspergillus PCR is limited by the extraction procedure and not by PCR amplification. For PCR testing of whole blood, it is essential that large blood volumes (≥3 ml) be efficiently lysed before bead beating to disrupt the fungal cell and performance of an internal control PCR to exclude false negativity. DNA should be eluted in volumes of <100 μl.
Early diagnosis and prompt initiation of specific antifungal treatment are essential for improving the prognosis of mucormycosis. We aimed to assess the performance of serum Mucorales quantitative polymerase chain reaction (qPCR) for the early diagnosis and follow-up of mucormycosis.We prospectively enrolled 232 patients with suspicion of invasive mold disease, evaluated using standard imaging and mycological procedures. Thirteen additional patients with proven or probable mucormycosis were included to analyze DNA load kinetics. Serum samples were collected twice-a-week for Mucorales qPCR tests targeting the Mucorales genera Lichtheimia, Rhizomucor, and Mucor/Rhizopus.The sensitivity was 85.2%, specificity 89.8%, and positive and negative likelihood ratios 8.3 and 0.17, respectively in this prospective study. The first Mucorales qPCR-positive serum was observed a median of 4 days (interquartile range [IQR], 0-9) before sampling of the first mycological or histological positive specimen and a median of one day (IQR, -2 to 6) before the first imaging was performed. Negativity of Mucorales qPCR within seven days after liposomal-amphotericin B initiation was associated with an 85% lower 30-day mortality rate (adjusted hazard ratio = 0·15, 95% confidence interval [.03-.73], P = .02).Our study argues for the inclusion of qPCR for the detection of circulating Mucorales DNA for mucormycosis diagnosis and follow-up after treatment initiation. Positive results should be added to the criteria for the consensual definitions from the European Organization for the Research and Treatment of Cancer/Mycoses Study Group Education and Research Consortium (EORTC/MSGERC), as already done for Aspergillus PCR.
Abstract Background. Coelomycetes are rarely but increasingly reported in association with human infections involving mostly skin and subcutaneous tissues, both in immunocompetent and immunocompromised patients. Coelomycetes constitute a heterogeneous group of filamentous fungi with distinct morphological characteristics in culture, namely an ability to produce asexual spores within fruit bodies. Methods. We included all cases of proven primary cutaneous and/or subcutaneous infections due to coelomycetes received for identification at the French National Reference Center for Invasive Mycoses and Antifungals between 2005 and 2014. Eumycetoma, chromoblastomycosis, and disseminated infections were excluded. Results. Eighteen cases were analyzed. The median age was 60.5 years. In all cases, patients originated from tropical or subtropical areas. An underlying immunodepression was present in 89% of cases. Cutaneous and/or subcutaneous lesions, mainly nodules, abscesses, or infiltrated plaques, were observed in distal body areas. Isolates of different genera of coelomycetes were identified: Medicopsis (6), Paraconiothyrium (3), Gloniopsis (3), Diaporthe (3), Peyronellaea (2), Lasiodiplodia (1). Lesion treatment consisted of complete (10) or partial (2) surgical excision and/or the use of systemic antifungal therapy, namely voriconazole (5) and posaconazole (4). Literature review yielded 48 additional cases of cutaneous and/or subcutaneous infections due to coelomycetes. Conclusions. Infectious diseases physicians should suspect coelomycetes when observing cutaneous and/or subcutaneous infections in immunocompromised hosts from tropical areas; a sequence-based approach is crucial for strains identification but must be supported by consistent phenotypic features; surgical treatment should be favored for solitary, well limited lesions; new triazoles may be used in case of extensive lesions, especially in immunocompromised patients.
ABSTRACT A cytochrome b ( cytb ) gene quantitative PCR (qPCR) assay was developed to diagnose malaria in travelers. First, manual and automated DNA extractions were compared and automated DNA extraction of 400 μl of blood was found to be more efficient. Sensitivity was estimated using the WHO international standard for Plasmodium falciparum DNA and compared to that of a previously published qPCR targeting the 18S rRNA coding gene (18S qPCR). The limit of detection of the cytb qPCR assay was 20 DNA copies (i.e., 1 parasite equivalent) per 400 μl of extracted whole blood and was comparable for the two qPCR assays. Both qPCR assays were used on blood samples from 265 consecutive patients seen for suspicion of malaria. There were no microscopy-positive and qPCR-negative samples. Positive cytb qPCR results were observed for 51 samples, and all but 1 were also 18S qPCR positive. Eight (16%) of these 51 samples were negative by microscopic examination. The 8 cytb qPCR-positive and microscopy-negative samples were from African patients, 3 of whom had received antimalarial drugs. Three non- P. falciparum infections were correctly identified using an additional qPCR assay. The absence of PCR inhibitors was tested for by the use of an internal control of mouse DNA to allow reliable quantification of circulating DNA. The high analytical sensitivity of both qPCR assays combined with automated DNA extraction supports its use as a laboratory tool for diagnosis and parasitemia determination in emergencies. Whether to treat qPCR-positive and microscopy-negative patients remains to be determined.
The diagnosis of congenital toxoplasmosis frequently relies on PCR tests of amniotic fluid (AF). A duplex real-time quantitative PCR test based on fluorescence resonance energy transfer was developed to quantify the parasite load and to decrease the risk of contamination. An internal control based on the detection of 10 pg mouse DNA added to the AF was included to check for PCR efficiency. The relationship between the parasite load and the occurrence of ultrasonographic abnormalities in 87 samples of AF was analyzed. Seven AF (8%) had a parasitic load > 10(3); 14 (16%) had > 10(2)-< or =10(3); 26 (30%) had > 10-< or = 10(2); and 40 (46%) had < or = 10 parasites/ml. Four of the six AF with cerebral ventriculomegaly had >10(3) parasites/ml. The other two had 130 and 24 parasites/ml, respectively. No parasitic loads of > 10(3) parasites/ml and no ultrasonographic abnormalities were observed in the 11 AF with maternal toxoplasmosis in the third trimester. Therefore, there is a trend to associate high parasite count with ultrasonographic abnormality, but the main concern remains early maternal infection. The importance of quantification should be better evaluated with postnatal studies. The duplex LightCycler PCR test currently provides rapid and safe results.
