Screening paracoccidioidomycosis by double immunodiffusion test in a referred diagnostic center in Brazilian southeastern: an accessible tool

Maifrede, Simone Bravim; Kruschewsky, Wdson Luís Lima; Patricio, Suzana Alves; Falqueto, Aloísio; Peçanha, Paulo Mendes; Malaquias, Luiz Cosme Cotta; Pôssa, Ana Paula; Camargo, Zoilo Pires de; Rodrigues, Anderson Messias; Gonçalves, Sarah Santos; Velloso, Tânia Regina Grão

doi:10.1007/s15010-021-01704-8

Cited by 5 publications

(8 citation statements)

References 24 publications

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“…Therefore, our data draws attention to the urgent need to expand the offer of serological diagnostic tests using antigenic preparations from P. lutzii ( Gegembauer et al 2014 , Queiroz Junior et al. 2014 , Maifrede et al. 2021 ) or the availability of PCR assays for the detection of P. lutzii DNA ( Pinheiro et al.…”

Section: Discussionmentioning

confidence: 99%

Trends in the molecular epidemiology and population genetics of emergingSporothrixspecies

Roberto¹,

Carvalho²,

Beale³

et al. 2021

Studies in Mycology

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Paracoccidioidomycosis (PCM) is a life-threatening systemic fungal infection acquired after inhalation of Paracoccidioides propagules from the environment. The main agents include members of the P. brasiliensis complex (phylogenetically-defined species S1, PS2, PS3, and PS4) and P. lutzii . DNA-sequencing of protein-coding loci (e.g., GP43 , ARF , and TUB1 ) is the reference method for recognizing Paracoccidioides species due to a lack of robust phenotypic markers. Thus, developing new molecular markers that are informative and cost-effective is key to providing quality information to explore genetic diversity within Paracoccidioides . We report using new amplified fragment length polymorphism (AFLP) markers and mating-type analysis for genotyping Paracoccidioides species. The bioinformatic analysis generated 144 in silico AFLP profiles, highlighting two discriminatory primer pairs combinations (#1 EcoRI-AC/MseI-CT and #2 EcoRI-AT/MseI-CT). The combinations #1 and #2 were used in vitro to genotype 165 Paracoccidioides isolates recovered from across a vast area of South America. Considering the overall scored AFLP markers in vitro (67–87 fragments), the values of polymorphism information content ( PIC = 0.3345–0.3456), marker index ( MI = 0.0018), effective multiplex ratio ( E = 44.6788–60.3818), resolving power ( Rp = 22.3152–34.3152), discriminating power ( D = 0.5183–0.5553), expected heterozygosity ( H = 0.4247–0.4443), and mean heterozygosity ( H avp = 0.00002–0.00004), demonstrated the utility of AFLP markers to speciate Paracoccidioides and to dissect both deep and fine-scale genetic structures. Analysis of molecular variance (AMOVA) revealed that the total genetic variance (65-66 %) was due to variability among P. brasiliensis complex and P. lutzii (PhiPT = 0.651–0.658, P < 0.0001), supporting a highly structured population. Heterothallism was the exclusive mating strategy, and the distributions of MAT1-1 or MAT1-2 idiomorphs were not significantly skewed (1:1 ratio) for P. brasiliensis s. str. (χ 2 = 1.025; P = 0.3113), P. venezuelensis (χ 2 = 0.692; P = 0.4054), and P. lutzii (χ 2 = 0.027; P = 0.8694), supporting random mating within each species. In contrast, skewed distributions were found ...

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Section: Discussionmentioning

confidence: 99%

Trends in the molecular epidemiology and population genetics of emergingSporothrixspecies

Roberto¹,

Carvalho²,

Beale³

et al. 2021

Studies in Mycology

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“…Though many validated methods for detecting anti- Paracoccidioides serum antibodies exist, most are conducted only in research centers. There also remains significant limitations, owing to cross-reactivity with other infectious fungi, such as Histoplasma and Aspergillus [ 25 , 99 , 109 ].…”

Section: Diagnosis Of Paracoccidioidomycosismentioning

confidence: 99%

“…By evaluating different antigenic preparations from P. lutzii using the immunodiffusion technique, Gegembauer et al [ 112 ] demonstrated that tests employing P. brasiliensis antigens might yield false-negative results when P. lutzii is the causative agent [ 109 , 119 ]. Maifrede et al [ 109 ] showed that 7 of 21 sera samples negative for P. brasiliensis antigen were positive for P. lutzii when the Pb339 exoantigen and PIEPM208 CFA were applied. We can infer that the frequency of P. lutzii may be higher than reported in endemic areas because gp43 is the most commonly used antigen in routine laboratory examinations [ 9 , 77 , 109 ].…”

Section: Diagnosis Of Paracoccidioidomycosismentioning

confidence: 99%

“…Maifrede et al [ 109 ] showed that 7 of 21 sera samples negative for P. brasiliensis antigen were positive for P. lutzii when the Pb339 exoantigen and PIEPM208 CFA were applied. We can infer that the frequency of P. lutzii may be higher than reported in endemic areas because gp43 is the most commonly used antigen in routine laboratory examinations [ 9 , 77 , 109 ].…”

Section: Diagnosis Of Paracoccidioidomycosismentioning

confidence: 99%

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Paracoccidioidomycosis: What We Know and What Is New in Epidemiology, Diagnosis, and Treatment

Peçanha

Peçanha-Pietrobom

Velloso

et al. 2022

JoF

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Paracoccidioidomycosis (PCM) is a systemic mycosis endemic to Latin America caused by thermodimorphic fungi of the genus Paracoccidioides. In the last two decades, enhanced understanding of the phylogenetic species concept and molecular variations has led to changes in this genus’ taxonomic classification. Although the impact of the new species on clinical presentation and treatment remains unclear, they can influence diagnosis when serological methods are employed. Further, although the infection is usually acquired in rural areas, the symptoms may manifest years or decades later when the patient might be living in the city or even in another country outside the endemic region. Brazil accounts for 80% of PCM cases worldwide, and its incidence is rising in the northern part of the country (Amazon region), owing to new settlements and deforestation, whereas it is decreasing in the south, owing to agriculture mechanization and urbanization. Clusters of the acute/subacute form are also emerging in areas with major human intervention and climate change. Advances in diagnostic methods (molecular and immunological techniques and biomarkers) remain scarce, and even the reference center’s diagnostics are based mainly on direct microscopic examination. Classical imaging findings in the lungs include interstitial bilateral infiltrates, and eventually, enlargement or calcification of adrenals and intraparenchymal central nervous system lesions are also present. Besides itraconazole, cotrimoxazole, and amphotericin B, new azoles may be an alternative when the previous ones are not tolerated, although few studies have investigated their use in treating PCM.

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“…From a serological perspective, it is not uncommon to find patients with negative serological results for the B-339 antigen in double immunodiffusion assay but positive in direct mycological examination [ 5 , 57 , 58 , 59 , 60 , 61 ]. In these paradoxical cases, identifying P. lutzii may be essential to recognizing false negative cases in serology and mapping the areas where regional antigens from P. lutzii are needed.…”

Section: Discussionmentioning

confidence: 99%

Development of a Multiplex qPCR Assay for Fast Detection and Differentiation of Paracoccidioidomycosis Agents

Pinheiro

Pôssa

Ricci³

et al. 2023

JoF

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Classic paracoccidioidomycosis (PCM) is a potentially deadly neglected tropical systemic mycosis caused by members of the Paracoccidioides brasiliensis complex (P. brasiliensis s. str., P. americana, P. restrepiensis, and P. venezuelensis) and P. lutzii. The laboratorial diagnosis of PCM relies on observing pathognomonic structures such as the “steering wheel” or “Mickey Mouse” shape in the direct mycological examination, fresh biopsied tissue in 10% KOH, histopathological analysis, and/or the isolation of the fungus in culture. However, these procedures are time-consuming and do not allow for the speciation of Paracoccidioides due to overlapping morphologies. Here, we propose a new one-tube multiplex probe-based qPCR assay to detect and recognize agents of the P. brasiliensis complex and P. lutzii. Primers (Paracoco-F and Paracoco-R) and TaqMan probes (PbraCx-Fam, Plu-Ned, and Paracoco-Vic) were developed to target the rDNA (ITS2/28S) in the Paracoccidioides genome. A panel of 77 Paracoccidioides isolates revealed a 100% specificity (AUC = 1.0, 95% CI 0.964–1.000, p < 0.0001) without cross-reacting with other medically relevant fungi or human and murine DNA. The lower limit of detection was 10 fg of gDNA and three copies of the partial rDNA amplicon. Speciation using qPCR was in perfect agreement with AFLP and TUB1-RFLP markers (kappa = 1.0). As a proof of concept, we assessed a panel of 16 formalin-fixed and paraffin-embedded specimens from histopathologically confirmed PCM patients to reveal a significant sensitivity of 81.25% and specificity of 100% (AUC = 0.906 ± 0.05, 95% CI = 0.756–0.979, p < 0.0001, Youden index J = 0.8125). Our assay achieved maximum sensitivity (100%) and specificity (100%) using fresh clinical samples (n = 9) such as sputum, bronchoalveolar lavage, and tissue fragments from PCM patients (AUC = 1.0, 95% CI 0.872–1.000, p < 0.0001, Youden index J = 1.0). Overall, our qPCR assay simplifies the molecular diagnosis of PCM and can be easily implemented in any routine laboratory, decreasing a critical bottleneck for the early treatment of PCM patients across a vast area of the Americas.

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Screening paracoccidioidomycosis by double immunodiffusion test in a referred diagnostic center in Brazilian southeastern: an accessible tool

Cited by 5 publications

References 24 publications

Trends in the molecular epidemiology and population genetics of emergingSporothrixspecies

Trends in the molecular epidemiology and population genetics of emergingSporothrixspecies

Paracoccidioidomycosis: What We Know and What Is New in Epidemiology, Diagnosis, and Treatment

Development of a Multiplex qPCR Assay for Fast Detection and Differentiation of Paracoccidioidomycosis Agents

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