Diagnosis of common dermatophyte infections by a novel multiplex real-time polymerase chain reaction detection/identification scheme

BACKGROUND: In the absence of a functional dermatophyte-specific polymerase chain reaction (PCR), current diagnosis of dermatophytoses, which constitute the commonest communicable diseases worldwide, relies on microscopy and culture. This combination of techniques is time-consuming and notoriously low in sensitivity. OBJECTIVES: Recent dermatophyte gene sequence records were used to design a real-time PCR assay for detection and identification of dermatophytes in clinical specimens in less than 24 h. PATIENTS AND METHODS: Two assays based on amplification of ribosomal internal transcribed spacer regions and on the use of probes specific to relevant species and species-complexes were designed, optimised and clinically evaluated. One assay was for detecting the Trichophyton mentagrophytes species complex plus T. tonsurans and T. violaceum. The second assayed for the T. rubrum species complex, Microsporum canis and M. audouinii. RESULTS: The analytical sensitivity of both assays was 0.1 pg DNA per reaction, corresponding to 2.5-3.3 genomes per sample. The protocol was clinically evaluated over 6 months by testing 92 skin, nail and hair specimens from 67 patients with suspected dermatophytosis. Real-time PCR detected and correctly identified the causal agent in specimens from which T. rubrum, T. interdigitale, M. audouinii or T. violaceum grew in culture, and also identified a dermatophyte species in an additional seven specimens that were negative in microscopy and culture. CONCLUSIONS: This highly sensitive assay also proved to have high positive and negative predictive values (95.7% and 100%), facilitating the accurate, rapid diagnosis conducive to targeted rather than empirical therapy for dermatophytoses.     

Molecular diagnosis of cutaneous leishmaniasis and species identification: analysis of 122 biopsies with varied parasite index

Background: Cutaneous leishmaniasis is endemic in the Middle East and North Africa. Confirming the diagnosis histologically depends on amastigote identification, which varies significantly depending on the inoculum, strain type, host response and disease stage. Accurate histological diagnosis is mandatory for appropriate therapy. Methods: Skin biopsies from 122 patients from Lebanon, Syria and Saudi Arabia with clinical diagnosis of untreated leishmaniasis were reviewed and clinical data extracted. Cases were classified according to the modified Ridley's parasitic index. DNA was extracted from formalin‐fixed paraffin‐embedded blocks. Polymerase chain reaction (PCR) was performed using Leishmania‐specific ribosomal internal transcribed spacer 1 (ITS1‐PCR). Nested ITS1‐PCR was performed on cases negative for conventional ITS1‐PCR. ITS1‐PCR amplicons were digested with HaeIII for subsequent restriction fragment length polymorphism (RFLP) subspeciation. Results: Of 122 cases, 54 (44.3%) showed a parasitic index of 0–1+ (no unequivocal amastigotes). ITS1‐PCR (conventional and nested) was positive for all cases as compared with negative control tissue. RFLP identified Leishmania tropica in all cases. Patients with clinically suspected leishmaniasis, whose skin biopsies failed to detect amastigotes represented 44.3% of our cases. Conclusions: In this study, we describe a rapid and optimized protocol from DNA extraction to leishmaniasis subspeciation. ITS1‐PCR showed high sensitivity and specificity in confirming clinically suspected cases. Yehia L, Adib‐Houreih M, Raslan WF, Kibbi A‐G, Loya A, Firooz A, Satti M, El‐Sabban M, Khalifeh I. Molecular diagnosis of cutaneous leishmaniasis and species identification: analysis of 122 biopsies with varied parasite index.     

聚合酶链反应检测麻风分枝杆菌的研究进展

麻风的早期诊治对减少疾病传播、预防疾病进展和致残起重要作用。本文对PCR技术在检测麻风杆菌中的应用作一综述。     

Molecular detection of dermatophytes and nondermatophytes in onychomycosis by nested polymerase chain reaction based on 28S ribosomal RNA gene sequences

Background  Onychomycosis is often caused by dermatophytes, but the role of nondermatophytes is underestimated due to the difficulty of identifying them by conventional direct microscopy and culture.
Objectives  This study aims to detect nondermatophytes, as well as dermatophytes, in the nail samples of patients with onychomycosis using a polymerase chain reaction (PCR)-based culture-independent method.
Materials and methods  The nested PCR assay targeting the sequence of the 28S ribosomal RNA gene was used to amplify fungal DNAs from 50 microscopy-positive nail specimens. Newly designed primer sets for dermatophyte universal, Trichophyton rubrum , T. mentagrophytes , Aspergillus spp., Scopulariopsis brevicaulis , Fusarium solani , F. oxysporum , F. verticillioides , Candida albicans and C. tropicalis were used after confirmation of their specificity.
Results  Forty-seven cases (94%) were positive for fungal DNA, among which dermatophytes were detected in 39 cases (83·0%): T. rubrum in 35 cases (74·5%) and T. mentagrophytes in eight cases (17·0%). Surprisingly, nondermatophytes were detected in 18 cases (38·3%), both dermatophytes and nondermatophytes in 10 cases (21·3%) and nondermatophytes alone in eight cases (17·0%). Aspergillus spp. alone was observed in five cases (10·6%).
Conclusions  This study indicates that most of the affected nail plates of patients with onychomycosis were positive for specific fungal DNAs, and suggests that nondermatophytes detected at high rates may be involved in the pathogenesis of onychomycosis.     

Fast and sensitive detection of Trichophyton rubrum DNA from the nail samples of patients with onychomycosis by a double-round polymerase chain reaction-based assay

BACKGROUND: Trichophyton rubrum is one of the most frequently isolated pathogens in onychomycosis. Isolation of T. rubrum from nail samples by traditional methods is time-consuming and has a high false-negative rate of detection. OBJECTIVES: To investigate the detection of T. rubrum in nail samples using DNA detection methods. METHODS: A total of 62 nail samples from onychomycosis patients with T. rubrum infection were evaluated by culture on Sabouraud's dextrose agar plus chloramphenicol, cycloheximide and gentamicin and compared with genotyping methods utilizing DNA extracted directly from nails. Trichophyton rubrum DNA isolated directly from nails was amplified using two different conserved regions [actin gene and internal transcribed spacer 1 (ITS)] in double-round polymerase chain reaction (PCR) assays. RESULTS: Forty-eight of 62 (77.4%) samples were potassium hydroxide (KOH) positive, but T. rubrum culture was positive in only 14 of 62 (22.6%) samples. By contrast, direct T. rubrum DNA detection rate was 59.7% (37/62) by actin gene and 45.2% (28/62) by ITS1 region PCR assays corresponding to higher detection frequencies compared with culture with P < 0.001 and < 0.008, respectively. The combined detection of actin and ITS1 was 69.4% (43/62). Interestingly, T. rubrum DNA was detected in 9 out of 14 (64.3%) of KOH- and culture-negative samples. Importantly, 15 culture-negative samples collected from patients undergoing antifungal treatment tested PCR positive using the actin region. CONCLUSIONS: These results suggest that a direct DNA detection protocol is more sensitive, accurate and faster than traditional culture-based methods. It can be useful to detect T. rubrum in patients undergoing antifungal therapy and who have been reported mycologically cured on the basis of a culture-based method.     

PCR直接检测皮肤分枝杆菌感染的研究

目的 探讨PCR直接检测皮肤分枝杆菌感染作为诊断的可能性.方法 PCR检测方法及培养方法(Löwenstein-Jensen培养基)比较分枝杆菌纯菌悬液、模拟皮肤分枝杆菌感染标本前处理前后、疑似皮肤分枝杆菌感染的临床标本前处理前后的检测结果.结果 分枝杆菌纯菌悬液的PCR法和培养法敏感性皆为1×10²个菌细胞/mL.模拟临床标本在经过前处理后在浓度为1×10²个菌细胞/mL的数量级上PCR检测阳性率为60%,而未经过前处理的模拟临床标本在此数量级上PCR检测阳性率为0,培养方法的阳性率为100%,但在浓度为1×10³个菌细胞/mL数量级上PCR检测阳性率为100%.37例临床疑似皮肤分枝杆菌感染标本经前处理后,PCR检测7例阳性,而未经前处理的标本同法检测2例阳性,但培养方法可检出9例阳性.结论 临床皮肤标本经前处理后,PCR检测的敏感性已接近80%,但能明显缩短检测时间.     

Unusual histopathological features of cutaneous leishmaniasis identified by polymerase chain reaction specific for Leishmania on paraffin-embedded skin biopsies

BACKGROUND: Cutaneous leishmaniasis (CL) is rare in Northern Europe and may be overlooked because colleagues have little experience with it. OBJECTIVES: To identify manifestations of CL that may escape diagnosis. METHODS: Correlation of clinical diagnosis and histopathological findings in 28 biopsy specimens taken from 19 patients with CL confirmed by polymerase chain reaction (PCR) specific for Leishmania. RESULTS: In only one patient was the clinical diagnosis CL; other diagnoses included: malignant epithelial neoplasms (5), follicular cyst (2), atypical mycobacteriosis (1), sarcoidosis (2) and lymphoma (1). Lesions were single (15) or few (4) nodules predominantly situated on the extremities or face (16). Histopathological findings were diagnostic of CL in only 10 cases. In nine cases Leishmania was not identified microscopically; histopathological diagnoses were: granulomatous dermatitis (6), lupoid rosacea (1), foreign body granuloma (1) and granuloma annulare (1). Unaltered epidermis (9), nodular infiltrates (5), numerous multinucleated histiocytes (3), palisaded granulomas with fibrinoid centres (2), sarcoidal granulomas (4) and elastophagocytosis (1) misled the histopathologists in these cases. CONCLUSIONS: CL seems often to be misdiagnosed clinically in countries where it is not endemic. Histopathologically, CL may be misinterpreted as sarcoidosis, foreign body granuloma, lupoid rosacea and granuloma annulare, especially when Leishmania is not seen microscopically. We suggest that in Northern Europe, PCR for Leishmania-specific DNA should be performed routinely in any granulomatous dermatitis presenting as a single or few nodules on the extremities or face, even when a diagnosis of CL was not considered by the referring clinician.     

聚合酶链反应与血清酶联免疫吸附试验在生殖器疱疹中的应用对比分析

目的:考察聚合酶链反应(PCR)与血清酶联免疫吸附试验(ELISA)在生殖器疱疹中的应用效果差异。方法:选取140例生殖器皮肤、黏膜损伤患者,分别采用聚合酶链反应与血清酶联免疫吸附试验检测标本中单纯疱疹病毒,对两种结果不符患者采用第二种PCR进行检测。结果:140例样本中,PCR检测出阳性样本59例,单纯HSV-1感染患者6例,单纯HSV-2感染患者46例,混合感染患者7例。ELISA检出阳性样本57例。140例样本中,有17例结果不符,采用第二种PCR进行检查证实,ELISA检测法敏感性为96.3%、特异性为98.8%、阳性预测值为89.8%。PCR法敏感性为97.1%、特异性为92.8%、阳性预测值为95.6%。结论:相比于PCR法,ELISA法具有更高的敏感度和特异性,避免了样本间的相互污染,具有临床应用价值。     

A case of zoster sine herpete presenting with thoracic radicular pain diagnosed by polymerase chain reaction in skin exudate

Varicella zoster virus (VZV) can cause radicular pain in the absence of skin lesions; such cases are referred to as zoster sine herpete (ZSH) and are usually diagnosed by using serological assays or polymerase chain reaction (PCR). An effort is underway to detect VZV DNA in novel specimens rather than conventional samples (e.g., blood or cerebrospinal fluid) for PCR. There are two reports that PCR analysis in the exudate of the auricular skin can be a useful diagnostic tool for the diagnosis of ZSH in patients presenting with cranial nerve paralysis without herpetic eruptions. Here, we report a case of ZSH diagnosed by using PCR analysis of skin exudates in a patient who developed thoracic radicular pain. This is believed to be the first case of ZSH diagnosed using PCR analysis of skin exudate in a patient in whom the cranial nerve was not involved.     

Detection of herpesvirus DNA in peripheral blood mononuclear cells and skin lesions of patients with pemphigus by polymerase chain reaction

Pemphigus is an autoimmune disease where both endogenous (genetic) and exogenous (environmental) factors play a part. Viral infections, in particular herpesvirus infections, have been identified as a possible triggering factor for pemphigus. In this study, using the polymerase chain reaction, we studied peripheral blood mononuclear cells (PBMC) and skin biopsies from patients with pemphigus, and in some of these were able to demonstrate the presence of DNA sequences of herpes simplex virus 1/2 (50% in PBMC and 71% in skin biopsies), Epstein-Barr virus (15% in PBMC and 5% in skin biopsies) and human herpesvirus 6 (20% in PBMC only). However, the inability to detect herpesvirus DNA consistently in these cases suggests that viral infection may only be an occasional factor triggering the outbreak or exacerbation of the disease. The possible role of interferons and interleukins in the pathogenesis of virus-induced pemphigus is discussed.     

First evaluation of polymerase chain reaction assays used for diagnosis of Mycoplasma genitalium in Russia

Background  Diagnosis of Mycoplasma genitalium is entirely based on nucleic acid amplification tests (NAATs). In Russia, several M. genitalium polymerase chain reaction (PCR) assays have been developed; however, any evaluation of their performance has never been performed.
Objective  To assess the performance of five PCRs developed and currently used in Russia for diagnosis of M. genitalium .
Materials and methods  Vaginal swabs and first voided urine samples (FVUs) from 281 females and urethral swabs and FVUs from 125 males were analysed using three conventional PCRs and two real-time PCRs developed by three Russian companies. As reference tests, a real-time PCR targeting the MgPa adhesin gene was used; positive results were confirmed by two conventional PCRs targeting the 16S rRNA gene and MgPa gene, respectively. For evaluation of detection limits and analytical specificities, a blinded control panel consisting of dilutions of six strains of M. genitalium and 14 other Mycoplasma species was tested.
Results  The prevalence of M. genitalium was 2.5% among females and 9.6% among males. The highest sensitivity (71.4–100% in different specimens) was exhibited by one real-time PCRs. Conventional PCRs from two manufacturers failed to detect M. genitalium in any of the seven positive female FVUs. All tests had a 100% clinical specificity; however, one cross-reacted with Mycoplasma pneumoniae .
Conclusions  Only one of the five Russian PCRs displayed reasonable sensitivity for all specimen types, but the specificities of all assays were high. Accordingly, improvements regarding sensitivity of all the tests are needed. However, larger studies, including other populations, evaluating these assays are crucial.     

抗原捕获聚合酶链反应分型检测妇女生殖器单纯疱疹病毒

目的 :　建立直接分型检测妇女生殖器单纯疱疹病毒 (HSV)的抗原捕获聚合酶链反应 (AC -PCR)。方法 :　用抗HSV型共同性糖蛋白单克隆抗体 ,包被聚苯乙烯离心管 ,捕获HSV ,同时加入 3个引物 :HSV - 1 HSV - 2型共同性上游引物及HSV - 1和HSV - 2型特异性下游引物 ,进行PCR扩增。结果 :HSV - 1和HSV - 2标准病毒株均分别扩增出与设计大小相符的 4 77bp和 399bpDNA条带。AC -PCR可检测到 10PFUHSV - 1和 1PFUHSV - 2。用AC -PCR检测了 36 5份妇女生殖器拭子标本 ,阳性 10 1例(2 7.7% ) ,2 3例为HSV - 1(占 2 2 .8% ) ,78例为HSV - 2 (占 77.2 % ) ;其中 112份标本同时用AC -PCR和分离培养法检测 ,AC -PCR的阳性率为 2 6 .8% (30 112 ) ,分离培养法的阳性率为 2 0 .5 % (2 3 112 ) ,两者差异有显著性 (χ2 =4 .5 ,P <0 .0 5 )。结论 :　AC -PCR是特异、敏感、快速分型检测妇女生殖器HSV感染的方法     

Isolation and polymerase chain reaction typing of Borrelia afzelii from a skin lesion in a seronegative patient with generalized ulcerating bullous lichen sclerosus et atrophicus

A 64-year-old woman presented with bullous and ulcerating lichen sclerosus et atrophicus (LSA) on the neck, trunk, genital and perigenital area and the extremities. Histology of lesional skin showed the typical manifestations of LSA; in one of the biopsies spirochaetes were detected by silver staining. Despite treatment with four courses of ceftriaxone with or without methylprednisone for up to 20 days, progression of LSA was only stopped for a maximum of 1 year. Spirochaetes were isolated from skin cultures obtained from enlarging LSA lesions. These spirochaetes were identified as Borrelia afzelii by sodium dodecyl sulphate--polyacrylamide gel electrophoresis and polymerase chain reaction (PCR) analyses. However, serology for B. burgdorferi sensu lato was repeatedly negative. After one further 28-day course of ceftriaxone the lesions stopped expanding and sclerosis of the skin was diminished. At this time cultures for spirochaetes and PCR of lesional skin for B. afzelii DNA remained negative. These findings suggest a pathogenetic role for B. afzelii in the development of LSA and a beneficial effect of appropriate antibiotic treatment.     

Human herpesvirus 7 detection by quantitative real time polymerase chain reaction in primary cutaneous T-cell lymphomas and healthy subjects: lack of a pathogenic role

Background Primary cutaneous T‐cell lymphomas (CTCLs) are a heterogeneous group of lymphomas where the tumour population emerges within a multiple subclone pattern. Mycosis fungoides (MF) and Sézary syndrome (SS) are characterized by the expansion of clonal CD4+/CD45RO+ memory T cells. Lymphomatoid papulosis (LyP) is a chronic, lymphoproliferative disorder included in the CD30+ primary CTCL spectrum. Several studies have suggested a role of viral infection for super‐antigenic activation of T lymphocytes; however, evidence of their association with CTCLs is still lacking. Human herpesvirus (HHV) 7 is a CD4+ T‐lymphotropic herpesvirus; its restricted cellular tropism and the ability to induce cytokine production in infected cells could make it an important pathogenic cofactor in lymphoproliferative disorders. Objectives To investigate the presence of HHV7 DNA on CTCL and healthy skin donors (HD). Methods We used quantitative real time polymerase chain reaction to evaluate the potential pathogenic role of HHV7. Results Twenty‐seven of 84 (32·1%) HD were positive for HHV7 DNA. Twenty‐one of 148 (14·2%) patients with CTCLs were positive for HHV7 DNA: nine of 39 (23·1%) SS, six of 14 (42·9%) CD30+ CTCLs and six of 24 (25·0%) LyP, and HHV7 DNA was negative in all 71 patients with MF. Conclusions These results seem to exclude a pathogenic role of HHV7 in CTCLs, suggesting the possibility of skin as a latency site.     

Examination of mycosis fungoides for the presence of Epstein–Barr virus and human herpesvirus-6 by polymerase chain reaction

BACKGROUND: The aetiology of cutaneous T-cell lymphoma (CTCL) remains unknown despite numerous investigations. In recent years, retroviruses and human herpesviruses have been implicated to play a causal part in CTCL. OBJECTIVE: The aim of this study was to elucidate the possible aetiopathogenetic role of human herpesviruses (HHV) in mycosis fungoides (MF). METHODS: Polymerase chain reaction was used to study formalin-fixed, paraffin-embedded lesional skin biopsies from 92 subjects with MF to evidence possible presence of Epstein-Barr virus (EBV) and HHV-6. RESULTS: Biopsy specimens from nine subjects (9.8%) evidenced EBV DNA, whereas all except one of the subjects (1.1%) lacked HHV-6 DNA. CONCLUSIONS: Although these findings do not support a primary aetiological role for EBV and HHV-6 in classical CTCL, the possibility remains that both viruses, particularly EBV, may act as potential cofactors in the development of CTCL.     

Polymerase chain reaction in the diagnosis of onychomycosis: a large,single‐institute study

Summary Background  Tests commonly used in the diagnosis of onychomycosis include potassium hydroxide (KOH) preparation, histopathological examination with periodic acid–Schiff stain (PAS) and culture. These tests are either time‐consuming or require specially trained personnel. A recently developed polymerase chain reaction (PCR) assay has the potential to provide a quick, inexpensive, operator‐independent diagnosis of onychomycosis. Objective  To determine the range of fungal species detected by the PCR technique and to compare this technique with KOH, PAS and culture on patient specimens. Methods  A total of 176 dermatophytic and nondermatophytic fungal culture isolates were tested by PCR. Five hundred and fifty nail specimens from 550 patients with suspected onychomycosis were split and tested concurrently with PCR, PAS, KOH and culture. Results  PCR was positive in 65 out of 66 dermatophyte culture isolates and negative in all 110 nondermatophyte and yeast isolates. Overall, PAS, PCR, KOH and culture were positive in 54%, 37%, 40% and 22% of specimens, respectively. Fifty‐two per cent were positive for KOH and PCR. Conclusion  PCR is a specific, relatively sensitive test for onychomycosis. When used in conjunction with KOH, PCR can produce positivity rates similar to those with PAS alone.