Nine cases of Japan spotted fever diagnosed at our hospital in 2008

Background The diagnosis of Japan spotted fever (JSF) is very difficult in some cases. The initial diagnosis of JSF is very important to treat. Methods We report nine cases of Japan spotted fever (JSF) with variable clinical features diagnosed at our hospital in 2008. Results Concerning clinical symptom, the most frequent symptoms were fever (8/9) and erythema of the whole body (8/9), followed by eschar (4/9). Palmar erythema, vomiting, and headache were observed in two cases. Purpura and lymph node swelling were observed in one case. Complication with Disseminated intravascular coagulation (DIC) was observed in one case. Laboratory findings revealed elevated plasma level of C‐reactive protein (CRP) and liver dysfunction in all cases, and decreased platelet (7/9). Interestingly, all patients had a history of presumed infection in the Southern area of Miya River, where wild Japanese deer with ticks (vector of Rickettsia japonica) may reside. Conclusion Different procedures are performed to make a diagnosis of JSF. For an initial definite diagnosis and adequate treatment of JSF, PCR of samples taken from blood, and skin biopsy from erythema and eschar lesions are necessary. Paired serum to measure the titers of antibody against R. japonica is also important.     

Diagnosing leprosy: revisiting the role of the slit-skin smear with critical analysis of the applicability of polymerase chain reaction in diagnosis

Background Diagnosing leprosy is challenging, especially in early‐stage cases, and the need for a sensitive diagnostic tool is urgent. Polymerase chain reaction (PCR) holds promise as a simple and sensitive diagnostic tool, but its usefulness in the Indian context requires further evaluation. Slit‐skin smear (SSS) remains the conventional method of leprosy detection. Hence, this study was undertaken to evaluate and compare the diagnostic efficacy of PCR versus that of SSS. Methods Punch biopsy of skin and SSS were obtained from the active margins of lesions. Cases were clinically grouped according to whether they were multibacillary (MB) or paucibacillary (PB) and classified into tuberculoid (TT), borderline tuberculoid (BT), borderline lepromatous (BL), lepromatous (LL), histoid, and indeterminate groups after clinicopathological correlation. DNA was extracted from biopsy specimens, and multiplex PCR was carried out incorporating primers intended for the amplification of a specific 372‐bp fragment of a repetitive sequence of Mycobacterium leprae DNA. Results Among 164 patients, PCR was positive in 82.3%. The sensitivity of PCR was significantly greater (P < 0.0001) than that of SSS in both the MB (85.9% vs. 59.8%) and PB (75.4% vs. 1.8%) subgroups; the difference in sensitivity in the PB subgroup is remarkable. Positivity by PCR and SSS was found in 100% of LL and histoid leprosy, but PCR had significantly greater (P < 0.0001) positivity in BT leprosy and was of definite increased value in indeterminate and TT leprosy. Conclusions Polymerase chain reaction had higher sensitivity compared with SSS, especially in diagnostically challenging and PB cases. Thus, the use of this costly but sensitive tool should be restricted to this subgroup, because SSS is sufficiently sensitive in the diagnosis of LL and histoid leprosy.     

Cross‐sectional study of Treponema pallidum PCR in diagnosis of primary and secondary syphilis

Background

Syphilis remains a major challenge and a complex diagnosis. We aim to evaluate the role of polymerase chain reaction (PCR) in Treponema pallidum (Tp) detection in various types of biological samples in the diagnosis of early syphilis.

Methods

We conducted a cross‐sectional study including all attendees of the STI clinic with clinical suspicion of early syphilis. One or more specimens for the detection of Tp by PCR testing were collected.

Results

The overall sensitivity of Tp PCR test was 82.61% (95% CI: 68.6–92.2%). Tp PCR test had sensitivity of 84.6% (95% CI: 54.6–98.1%) in primary syphilis cases and 81.8% (95% CI: 64.5–93%) in secondary syphilis cases. PCR test performance was independent of HIV status.

Conclusion

Tp PCR test is a fast and reliable method for the detection of Tp in skin lesions of early syphilis, and it is a powerful tool in clinical settings.     

Molecular diagnosis of skin infections using paraffin‐embedded tissue – review and interdisciplinary consensus

Nucleic acid amplification techniques (NATs), such as PCR, are highly sensitive and specific methods that have become valuable supplements to culture and serology in the diagnosis of infectious disorders. However, especially when using formalin‐fixed and paraffin‐embedded tissue, these techniques are associated with both false‐negative and false‐positive results, a pitfall that is frequently misjudged. Representatives of the German Society of Hygiene and Microbiology (DGHM) and the German Society of Dermatology (DDG) therefore set out to develop a consensus – in the form of a review article – on the appropriate indications for NATs using paraffin‐embedded tissue, its contraindications, and the key points to be considered in the pre‐ and post‐analytical phase. Given that fresh, naive tissue is preferably to be used in the workup of a suspected infection, PCR analysis on paraffin sections represents an exception. The latter may be considered if an infection is suspected at a later point in time and fresh tissue has not been preserved or can no longer be obtained. Potential indications include confirmation of histologically suspected infections with Leishmania spp., Bartonella spp., Rickettsia spp., or in case of ecthyma contagiosum. Infections with, for example, mycobacteria or RNA viruses, on the other hand, are not considered useful indications for NATs using paraffin sections. In order to avoid misinterpretation of test results, it is essential that laboratory reports on NATs using paraffin‐embedded tissue contain information on the indication/diagnostic circumstances, the required and chosen pre‐analytical steps, the limitations of the method, and on diagnostic alternatives.     

Rapid identification of Sporothrix schenckii in biopsy tissue by PCR

Background The dimorphic fungus Sporothrix schenckii is the etiological agent of sporotrichosis, an important cutaneous mycosis with a worldwide distribution. At present, it is challenging to rapidly discover and identify Sporothrix schenckii in biopsy tissues nowadays. Aims To explore new methods for rapid diagnosis of sporotrichosis. Materials and Methods We screened specific primers for Sporothrix schenckii using 50 clinical isolates from patients with sporotrichosis. DNA was extracted from the lesions of 30 cases of clinically suspected sporotrichosis using the Graham s method of CTAB and amplified by PCR using the screened specific primers. Results The primer S2‐R2 was applicable for the identification of S. schenckii from different geographic areas and clinical types with high specificity and sensitivity. Twenty‐five out of the thirty cases (83.3%) amplified using the primer S2‐R2 showed positive bands. Further positive bands were observed in 95.6% of cases tested positive by fungal culture. Conclusions Using the PCR technique and specific primers, we developed a new diagnostic method that can rapidly diagnose sporotrichosis with tissues obtained from clinical biopsies.     

Transglutaminases as diagnostically relevant autoantigens in patients with gluten sensitivity

Background: Patients with gluten sensitivity, i. e. celiac disease and dermatitis herpetiformis have anti‐endomysial antibodies recognizing transglutaminases, which are usually detected on appropriate tissue sections. It would be desirable to have available a reliable, tissue‐independent serological diagnostic tool. We compared disease‐specificity and sensitivity of tTG versus eTG‐based detection systems for the diagnosis of anti‐endomysial IgA‐antibodies. Patients and Methods: We examined 204 serum samples in duplicates with commercial human ELISA‐kits: 54 healthy blood donors, 20 celiac disease, 29 dermatitis herpetiformis and 101 with other autoimmune dermatoses. Results: The tTG‐based ELISA proved to be very disease‐specific (100 %) and sensitive for the diagnosis of gluten sensitivity (95 % celiac disease; 96.6 % dermatitis herpetiformis). The eTG‐based ELISA was also perfectly specific (100 %), but only 15 % of celiac disease‐sera and 44.8 % of dermatitis herpetiformis‐sera yielded positive results. Conclusions: The human tTG‐ELISA fulfills all criteria of a screening test and, because of being investigator‐independent, inexpensive and highly reproducible, compares favorably with the current diagnostic gold standard (indirect immunofluorescence and biopsy) of celiac disease and dermatitis herpetiformis. The low sensitivity of the eTG‐ELISA may have technical reasons, but could theoretically also be linked to disease activity or indicate the existence of an as yet undefined disease subset. Studies are currently under way to address these issues.     

Development of a novel polymerase chain reaction–enzyme‐linked immunosorbent assay for the diagnosis of Trichophyton rubrum onychomycosis

Background  The prevalence of onychomycosis has increased steadily in the past decade. An accurate diagnosis at the outset is important for successful and cost‐effective treatment of patients. However, current diagnostic tests for onychomycosis are not rapid, sensitive or specific. Objectives  To develop a microsatellite‐based polymerase chain reaction (PCR)‐enzyme‐linked immunosorbent assay (MS‐ELISA) for the detection of Trichophyton rubrum, which is the most common aetiological agent of onychomycosis. Methods  An archival set of 434 nail and skin specimens from 217 patients was included as the test sample in this study. We also compared MS‐ELISA with an earlier published topoisomerase PCR‐ELISA (TI‐ELISA) using template DNA extracted by another method. Results  The MS‐ELISA detected the highest number of positive samples (69%) followed by direct microscopy (56%), TI‐ELISA (44%) and fungal culture (30%). When an identical DNA extraction method was applied to 120 specimens, the MS‐ELISA proved to be twice as sensitive as the TI‐ELISA. Conclusions  We have optimized a target gene and DNA extraction method for rapid detection of T. rubrum onychomycosis.     

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.     

Clinical utility of loop‐mediated isothermal amplification assay for the diagnosis of common alpha herpesvirus skin infections

Loop‐mediated isothermal amplification (LAMP) is a nucleic acid amplification method with a high specificity, efficiency and speed. No reports exist regarding the usefulness of LAMP for clinically suspected skin infections caused by herpes simplex virus (HSV) or varicella zoster virus (VZV). The aim of this study was to evaluate the clinical usefulness of LAMP in the diagnosis of common cutaneous alpha herpesvirus (HSV type 1 and 2, and VZV) infections. LAMP and real‐time polymerase chain reaction (PCR) were performed using swab samples collected from 106 patients with clinically suspected alpha herpesvirus skin infections. The results of LAMP performed with DNA extraction did not differ from those performed without DNA extraction. The sensitivity of LAMP tested against real‐time PCR was 96% in herpes simplex, 78% in eczema herpeticum, 93% in herpes zoster and 100% in varicella. No viral DNA was detected by LAMP in all negative real‐time PCR samples. Viral DNA load was significantly lower in samples with false‐negative LAMP results than in the LAMP‐positive samples. LAMP enables confirmation of clinically suspected cutaneous HSV and VZV infections. However, the sensitivity of LAMP is lower than real‐time PCR. The accuracy of LAMP may increase if sufficient viral DNA is obtained from lesions. LAMP performed without DNA extraction remains sensitive; thus, LAMP represents a quick and economical method for the diagnosis of common alpha herpesvirus skin infections.     

Introduction of a dermatophyte polymerase chain reaction assay to the diagnostic mycology service in Scotland

Background Dermatophytes are the major cause of superficial mycoses in samples submitted to Clinical Mycology, Glasgow. The most prevalent species is Trichophyton rubrum as identified classically by microscopy and culture. Recent advances in polymerase chain reaction (PCR) technology were examined for the feasibility of introducing a T. rubrum real‐time PCR assay into a routine diagnostic service. Objective To improve the diagnostic mycology service by the introduction of a real‐time PCR test for T. rubrum. Methods The DNA from 4972 nail and skin samples was obtained using the Qiagen QIAsymphony automated extractor. This DNA was subjected to real‐time PCR using T. rubrum‐specific primers and a probe. Results During phase 1 of the study, 862 samples were analysed; 446 of 470 specimens that grew T. rubrum were detected by PCR. Out of 4110 samples analysed during phase 2, 753 T. rubrum infections were diagnosed and reported within 72 h. A total of 3357 samples were negative for a fungal infection by PCR and microscopy; these were also reported within 72 h. Conclusions A vast reduction in the turnaround times can be achieved using this technique as opposed to classical methods. Samples which are PCR negative but microscopy positive are still subjected to culture. Screening samples for their suitability for PCR prior to processing eliminates the application of PCR for T. rubrum on inappropriate samples such those from the scalp or pityriasis versicolor.     

Molecular diagnostics of parapox virus infections

Background: The diagnosis of parapox virus infections relies primarily on a history of contact with infected animals. The clinical presentation is usually a non‐specific necrotic ulcer. The histology may also be non‐specific, especially with older lesions. Negative‐staining electron microscopy (EM) is a fast and reliable diagnostic tool, but is not widely available. Serological tests and the time‐consuming viral culture are also rarely used in Europe. Patients and methods: The diagnostic procedure in two patients with ecthyma contagiosum and milker's nodule using polymerase chain reaction specific for orthopox, parapox and Orf virus is explained. Diagnostics included bacterial culture, viral culture, histology and EM. In addition to these, a polymerase chain reaction (PCR) was performed in both cases. Results: The patient with ecthyma contagiosum was negative for ortho‐, parapox‐, and orf‐virus on PCR, whereas the patient with milker's nodule had a PCR positive for parapoxvirus. Conclusions: PCR is a simple, fast, and standardized method of diagnosis that can distinguish between the subgroups of parapoxviruses. A diagnosis can be made even in cases of ambiguous history or unspecific clinical presentation. The method is limited by the necessity to sample native material or to use neutrally buffered formalin in case of PCR from paraffin material.     

The significance of multiplex PCR/heteroduplex analysis-based TCR-γ gene rearrangement combined with laser-capture microdissection in the diagnosis of early mycosis fungoides

Background: The diagnosis of early mycosis fungoides (MF) is a big challenge to dermatologists and dermatopathologists because it lacks specific clinicopathologic features. Methods: Fifty‐two paraffin‐embedded skin samples from 50 patients, including 31 with suspected MF, 10 with typical MF and 9 with benign inflammatory dermatosis (BID), were obtained from our archives. DNA was extracted both by traditional phenol‐chloroform method and by the laser‐capture microdissection (LCM)‐proteinase K approach. The T_VG/T_JG, V_2–5/V_8–12/JGT₁ and BIOMED‐2‐TCR‐γ primers were used to assess TCR‐γ monoclonal rearrangement as measured by polymerase chain reaction (PCR). Results: In the suspected MF group, clonal TCR‐γ gene rearrangements were detected in 11/31 cases (35.5%) by phenol‐chloroform DNA extraction and in 25/31 cases (80.7%) by LCM‐proteinase K extraction (p < 0.05). While T‐cell clonality was detected in 8/10 cases (80%) by the phenol‐chloroform method and 10/10 cases (100%) by LCM (p > 0.05) in the typical MF group, no TCR‐γ monoclonal rearrangement was detected in the BID group. Conclusions: The strategy of multiple PCR/heteroduplex analysis for TCR‐γ gene rearrangement combined with LCM increases the detection rate of clonal TCR‐γ gene rearrangement in early MF cases and could provide strong evidence to confirm the diagnosis of early MF. Yang H, Xu C, Tang Y, Wan C, Liu W, Wang L. The significance of multiplex PCR/heteroduplex analysis‐based TCR‐γ gene rearrangement combined with laser‐capture microdissection in the diagnosis of early mycosis fungoides.     

Atypical presentation of Old-World cutaneous leishmaniasis, diagnosis and species identification by PCR

Background  The diagnosis of cutaneous leishmaniasis (CL) is traditionally based on microscopic demonstration of amastigote forms in tissue biopsies or smears. However, this method usually presents low sensitivity, and in atypical forms, CL may be overlooked because of similarity to other dermal diseases. Thus, it is necessary to apply specific diagnostic methods as polymerase chain reaction (PCR).
Objective  To evaluate the possible advantage of PCR in the diagnosis and species identification of CL in patients with atypical clinical presentation.
Methods  Fifty-one patients clinically suspected of CL with positive and negative controls were tested. After microscopic examination, extraction of DNA was performed on their smears and analysed by two specific PCR assays for diagnosis and species identification. For these methods, conserved and variable regions of kinetoplastic DNA (KDNA) of Leishmania species have been amplified, respectively. Atypical forms of CL were evaluated among PCR-positive patients.
Results  PCR results were positive in 37 out of 51 cases (72.5%), among whom microscopic examination revealed Leishmania amastigotes in only 3 (5.9%). Among these patients, 10 (27%) had atypical presentation of CL; using species-specific primers, 6 patients had Leishmania major , 3 had Leishmania tropica and 1 patient had no species diagnosis. None of the samples of other dermal diseases revealed positive results (specificity, 100%). All patients were successfully treated by CL-specific drug regimens.
Discussion  The results showed that KDNA PCR methods have a higher sensitivity compared with microscopic method. Moreover, PCR could identify the parasite species for specific therapy. Microscopic method had low sensitivity and less value in chronic and atypical CL cases.     

Comparison of fungal fluorescent staining and ITS rDNA PCR‐based sequencing with conventional methods for the diagnosis of onychomycosis

Background

The current gold standard for diagnosing onychomycosis is direct microscopic examination and culturing. Fungal culture is a time‐consuming procedure, while direct microscopy of potassium hydroxide (KOH) mounts suffers from low sensitivity. More rapid and sensitive methods for the diagnosis of onychomycosis are in high demand.

Objective

To establish an effective method for the diagnosis of onychomycosis by assessing the efficacies of fungal fluorescent staining and internal transcribed spacer (ITS) ribosomal DNA (rDNA) polymerase chain reaction (PCR)‐based sequencing.

Methods

A total of 204 clinical specimens from patients with suspected onychomycosis were analysed. The gold standard for a true positive sample was positive by KOH, culturing or both methods. All specimens were also tested by fungal fluorescent staining and ITS rDNA PCR‐based sequencing. We compared the detection, sensitivity and specificity for these two methods with conventional methods.

Results

In total, 126 (62%) and 102 (50%) were detected by fluorescent staining and PCR‐based sequencing, respectively. According to the conventional diagnostic standard, the sensitivity of fluorescent staining and PCR‐based sequencing was 97% and 78%, respectively, and specificities of 89% and 90%, respectively. Use of fluorescence enhanced the sensitivity of direct examination by 12% compared with KOH. PCR‐based sequencing increased the sensitivity by 6% compared with culturing.

Conclusions

Fluorescence microscopy has a higher sensitivity for the detection of fungi in nail specimens compared with KOH and can be used as a rapid screening tool. PCR‐based sequencing was faster and more sensitive compared with culture and when used in conjunction with fluorescence microscopy resulted in higher efficiency.     

In vivo confocal microscopy of basal cell carcinoma: a systematic review of diagnostic accuracy

Basal cell carcinoma (BCC) is the most prevalent type of skin cancer. Histologic analysis of punch biopsy or direct excision specimen is used to confirm clinical diagnosis. In vivo reflectance confocal microscopy (RCM) is a non‐invasive imaging modality that could facilitate early diagnosis and minimize unnecessary invasive procedures. We systematically reviewed diagnostic accuracy (sensitivity and specificity) of RCM in diagnosing primary BCCs to judge its usefulness. Eligible studies were reviewed for methodological quality using the QUADAS‐2 tool. We used the bivariate random‐effects model to calculate summary estimates of sensitivity and specificity. Six studies met the selection criteria and were included for analysis. The meta‐analysis showed a summary estimate of sensitivity 0.97 (95% CI, 0.90–0.99) and specificity 0.93 (95% CI, 0.88–0.96). All but one of the QUADAS‐2 items showed a high or unclear risk of bias with regards to patient selection. RCM may be a promising diagnostic tool, but the limited number of available studies and potential risk of bias of included studies do not allow us to draw firm conclusions. Future accuracy studies should take these limitations into account.     

Detection and identification of Trichophyton tonsurans from clinical isolates and hairbrush samples by loop‐mediated isothermal amplification system

Since the 1990s, there have been reports of the spread of dermatophytosis caused by Trichophyton tonsurans among contact sports athletes in several countries, including Japan. This study was performed to develop a loop‐mediated isothermal amplification (LAMP) system for rapid and accurate detection and identification of T. tonsurans from clinical isolates or hairbrush samples for diagnosis and to prevent the spread of infection. A specific primer set was prepared by comparing the whole genome sequence of T. tonsurans with those of six other closely related dermatophytes. After confirming the sensitivity and specificity of this system, LAMP assay was performed using 37 clinical samples obtained from three healthy volunteers and 24 judo athletes. A total of 155 fungal isolates (56 strains of various standard fungi, 96 identified T. tonsurans isolates, three hairbrush‐cultured isolates from judo athletes) and 37 hairbrush samples (34 samples from 24 judo athletes, and three samples from three healthy volunteers) were used for culture and LAMP assay, respectively. The assay showed no cross‐reactivity to standard strains other than T. tonsurans. The detection limit was 100 copies of DNA template per tube. All of the 96 T. tonsurans isolates were amplified, and all samples from healthy volunteers showed negative results. Four of the 34 hairbrush samples obtained from judo athletes showed positive results in LAMP assay, and two of the four were positive in both culture and LAMP assay. We developed a rapid LAMP system with high specificity and sensitivity for diagnosis of T. tonsurans infection.     

DETECTION OF MYCOBACTERIUM LEPRAE DNA IN FORMALIN-FIXED, PARAFFIN-EMBEDDED SAMPLES FROM MULTIBACILLARY AND PAUCIBACILLARY LEPROSY PATIENTS BY POLYMERASE CHAIN REACTION

Background. Diagnosis of paucibacillary leprosy is often difficult. A method that could confirm the diagnosis is the polymerase chain reaction (PCR) of M. leprae DNA. This reaction was applied to biopsied tissues of leprotic patients to determine the suitability and sensitivity of the reaction. Methods. Biopsy samples were taken from previously untreated patients with multibacillary (5 patients) and paucibacillary (3 patients) leprosy, fixed in formalin, and embedded in paraffin, DNA was extracted from paraffin blocks and PCR applied. The sensitivity of the PCR method was tested by using the serially diluted DNA sample as the template. Results. All eight patients showed a positive PCR for M. leprae DNA. The sensitivity was such that a single organism of M. leprae, as counted by staining of the acid-fast bacilli was identified by the PCR. Conclusions. The PCR method is simple, sensitive, specific, and does not require the use of radioisotopes. It can be applied to the unequivocal diagnosis of paucibacillary leprosy which is difficult by other means. The diagnosis can be obtained within 10 hours. Int J Dermatol 1993; 32:710–713     

Queensland tick typhus in rural New South Wales

We report five cases of Rickettsia australis infection from southern coastal New South Wales, Australia. All patients presented with a cutaneous eruption of erythematous papules and pustules and systemic features of malaise, headache, lymphadenopathy and myalgia. Acute kidney injury (AKI) was present in two of five cases and one of five cases had acute delirium. Improvement was only seen after treatment with doxycycline 100 mg b.i.d. Positive serology for R. australis was present in four of five cases and a positive polymerase chain reaction (PCR) was seen in one of five cases. Histology showed varying features, from neutrophilic vasculitis to Sweet's syndrome and lymphocytic vasculitis. Recent significant advances in the diagnosis of R. australis infection include an eschar swab or biopsy PCR and isolation of specific Rickettsia on serology. These investigations should be considered in the presence of any of the following features: eschar at site of a tick bite or lymphadenopathy and fever with an eruption of erythematous papules and pustules.     

A realistic approach to the sensitivity of PCR-DGGE and its application as a sensitive tool for the detection of clonality in cutaneous T-cell proliferations

Abstract The practical value of the detection of clonality within the T-cell receptor gamma locus by polymerase chain reaction for the diagnosis of cutaneous T-cell lymphomas is well known. However, studies dealing with this subject so far, with special emphasis on the sensitivity of the technique in comparison to, for example, Southern blotting have used mixtures of DNA in various concentrations instead of using mixtures of the cells involved, which would reflect the in vivo situation in a more realistic scope. The purpose of this study was therefore to determine the sensitivity and the limitations of the PCR assay by dilution experiments, using mixtures of cells. Furthermore we studied its applicability to cutaneous T-cell proliferative disorders. Two clonal T-cell lines (MyLa and Jurkat) served as positive control. Dilutions of MyLa cells, cultured normal human keratinocytes and peripheral blood mononuclear cells from lymphoma negative volunteers were used to assess the sensitivity of the PCR-DGGE assay. Skin samples from 4 patients with cutaneous T-cell lymphoma, 1 lesional lymph node, 2 blood samples from a patient with Sézary syndrome and 4 lymphoma-negative tissue samples were analysed. Two samples were uncertain for diagnosis of lymphoma. The PCR-DGGE assay consisted of a 2-round nested PCR with consensus primers within the TCR-gamma locus followed by electrophoretic separation of the product along a denaturing urea/formamide gradient gel. PCR-DGGE sensitivity was, to our knowledge, for the first time investigated for mixtures of lymphocytes (clonal and polyclonal) and keratinocytes. Clonal T-cells were detected in a concentration between 1–0.1% in keratinocytes, whereas the sensitivity was generally lower upon dilution in peripheral blood mononuclear cells or in a mixture of keratinocytes and peripheral blood mononuclear cells. Nevertheless, T-cell clonality was detected in 2 blood samples of a patient with Sézary syndrome, which were negative by Southern blot analysis. The crucial point of this work was the new approach to establish the sensitivity of the PCR-DGGE, in a way which more closely mimics the condition of clinical specimens. Instead of mixing and amplifying DNA extracted from clonal T-cell lines and polyclonal bone marrow cells, we amplified DNA from clonal and polyclonal cells which had been mixed in various ratios before DNA extraction. Polymerase chain reaction in conjunction with denaturing gradient gel electrophoresis is a sensitive and versatile molecular tool for the assessment of clonality of suspect cutaneous lesions. The determination of sensitivity using DNA extracted from premixed cells more closely corresponds to the actual test situation when testing skin samples.