Clinics in Dermatology (2011) 29, 548–556

Diagnostic procedures in dermatology

Eleonora Ruocco, MD, PhD a , Adone Baroni, MD, PhD a ,
Giovanna Donnarumma, PhD b , Vincenzo Ruocco, MD a,⁎
a
Department of Dermatology, Second University of Naples, via Sergio Pansini 5, 80131 Napoli, Italy
b
Department of Experimental Medicine, Second University of Naples, via Luigi de Crecchio 7, 80131 Napoli, Italy

Abstract Although most skin diseases can be diagnosed with simple visual inspection, laboratory
investigations are necessary in several clinical circumstances. This contribution highlights the usefulness
of routine diagnostic procedures that are often overlooked and the innovative methods of molecular
biology, which are expensive and require an experienced staff. Among the classic diagnostic
investigations are (1) the use of Wood's light in many dermatologic disorders (eg, vitiligo, pityriasis
versicolor, erythrasma, porphyrias), (2) cytodiagnosis of Tzanck in dermatologic practice (eg, herpetic
infections, molluscum contagiosum, leishmaniasis, pemphigus vulgaris, basal cell carcinoma,
erythroplasia of Queyrat, Hailey-Hailey disease), and (3) microscopic examination for fungal and
bacterial skin infections as well as for mite infestation using potassium hydroxide, simple saline, and
Gram stain. Modern molecular biotechnologies encompassing gene-specific polymerase chain reaction
and its variants have a substantial affect in selected cases of viral (especially herpes simplex virus),
bacterial, fungal, and protozoan (Leishmania) skin infections.
© 2011 Elsevier Inc. All rights reserved.

Introduction
The skin lends itself to diagnosis of its diseases more
readily than any other organ. With a clinically well-trained eye
and accurate patient history, most skin lesions can therefore be
diagnosed with simple visual inspection. There are, however,
clinical cases where the correct diagnosis can be made neither
at a glance nor after a prolonged, meticulous observation.
Among the several procedures and devices that can aid the
clinician in diagnosing nonimmediately recognizable cuta-
neous disorders, we first adopt those that grant no or low
invasiveness and rapid, reliable results at low cost. Of course,
when necessary, we do not hesitate to resort to more invasive,
sophisticated, and expensive laboratory investigations. As a
⁎ Corresponding author. Tel.: +39 081 566 68 30; fax: +39 081 546 87 59.
E-mail address: vincenzo.ruocco@unina2.it (V. Ruocco).
rule, an experienced clinician will from time to time find the
balance between advantages and disadvantages of the
disposable diagnostic aids and choose the more appropriate
one(s) for each patient. In this contribution, we show the
usefulness of some diagnostic devices and techniques that are
often overlooked in dermatologic practice.
Wood's light
The Wood's light (“black light”), first described in 1903,
is a source of ultraviolet light from which virtually all visible
rays have been excluded by a filter made of silicate with
nickel oxide (Wood's filter).1-4 Several types of Wood's
lamps are available, the most inexpensive ones being the
hand models with batteries and small fluorescent tubes. For
professional purposes, we recommend the use of a Wood's

0738-081X/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.clindermatol.2010.09.023
Diagnostic procedures in dermatology
light with a high-intensity 100-watt mercury vapor bulb and
power supply. We also suggest that one should always be
kept in the examination room where it will be readily
available and therefore used more often. The examiner
should use the Wood's light in a completely darkened room,
allow time for vision accommodation to see the contrasts
clearly, hold the lamp at least 10 cm from the skin, and warn
the patient against looking directly at the light source.
Possible applications of Wood's light in dermatologic
diagnosis are listed in Table 1.
Pigmentary disorders
Wood's light is strongly absorbed by melanin, which
makes this device a useful tool in the evaluation of
hyperpigmented or hypopigmented dyschromic skin lesions.
A lesion with an increased concentration of melanin in the
epidermis appears darker than surrounding normal skin
under Wood's light, which accounts for the usefulness of
Wood's light in determining the real borders of a lentigo
maligna.4 The same can be said for freckles, which become
more noticeable and in greater number when examined under
the Wood's light because of their epidermal pigmentation.1
This does not occur with an increased concentration of
melanin in the dermis. In fact, melasma, Mongolian spot,
nevus of Ota, and other lesions due to dermal pigmentation
show no enhancement of their darkness in the Wood's light
compared with visible light.
The Wood's lamp can be useful in distinguishing vitiligo
from pityriasis alba and nevus anemicus. Being due to loss of
epidermal melanin, the depigmented vitiligo lesions vividly
Table 1 Use of Wood’s light in dermatologic diagnosis
Pigmentary disorders
• Lentigo maligna
• Freckles
• Melasma
• Mongolian spot
• Nevus of Ota
• Vitiligo
• Pityriasis alba
• Nevus anemicus
• Tuberous sclerosis
Skin infections
• Tinea capitis
• Pityriasis versicolor
• Pityrosporum folliculitis
• Erythrasma
• Superinfected acne
• Pseudomonas pyocyanea infection
Porphyrias
• Porphyria cutanea tarda
• Congenital erythropoietic porphyria
• Erythropoietic protoporphyria
Tumors
• Squamous cell carcinoma
549
show as milk-white under Wood's light, which often allows
the clinician to discover many unnoticed early depigmented
areas. In pityriasis alba, mainly due to patchy parakeratosis
with only a little involvement of melanocyte activity, the ill-
defined white patches do not stand out under Wood's light.
In nevus anemicus, due to circumscribed dermal vasocon-
striction with normally pigmented overlying epidermis, the
pallor completely disappears under Wood's light.3 The ash
leaf-shaped white macules of tuberous sclerosis, often
overlooked during a clinical examination, can be easily
detected under Wood's light.
Skin infections
The importance of the Wood's light in the diagnosis of tinea
capitis has been well known for a long time. Hairs and scales
from tinea caused by the Microspora species exhibit bluish
green fluorescence due to pteridine, a metabolite produced by
the Microspora species. The fluorescence is dimmer in tinea
favosa, with a grayish green hue along the entire hair.
Tinea versicolor is the widest field of application of the
Wood's lamp. The actively infected areas show with a white,
yellowish to orange fluorescence evoking the shammy hue.
Under Wood's light, the infection often is found to be more
extensive than that seen with simple visual examination. The
Wood's lamp is particularly helpful in identifying the
localized forms involving the groin or a single antecubital
space in adults, the face, and the diaper area in infants. The
lamp is an exceedingly important tool to diagnose Pityros-
porum folliculitis, which can be regarded as the pure follicular
form of pityriasis versicolor.1 It is located chiefly on the upper
back, shoulders, and chest, usually mimicking a trivial acne,
although it is more papular than pustular. Under Wood's
light, the correct diagnosis of Pityrosporum folliculitis is
facilitated by a follicular bluish white fluorescence.
Erythrasma can be easily diagnosed by Wood's light
because of the brilliant coral-pink fluorescence due to the
presence of porphyrins produced by the causative bacterium. A
red fluorescence from pilosebaceous follicles can be detected in
acne lesions superinfected by Propionibacterium acnes, which
also produces porphyrins.3 The worsening of some forms of
acne in the summer (Mallorca acne) has been linked with the
photoactivation of porphyrins released in the pilosebaceous
follicles harboring P acnes. This photoactivation is thought to
be responsible for the subsequent inflammation.2
Pseudomonas pyocyanea infection, which is frequent in
patients with burns, can be recognized by the yellowish
green fluorescence that pyocyanin produces under Wood's
light. The Wood's lamp is used in burn centers to identify
Pseudomonas infections, not visible otherwise, hours before
cultures become positive.1,2
Porphyrias
Urine, feces, and occasionally, blister fluid fluoresce
pink-orange in porphyria cutanea tarda, teeth in
550 E. Ruocco et al.

congenital erythropoietic porphyria, and blood in eryth- Infections

ropoietic protoporphyria. 3
The clinical diagnosis of herpes simplex virus (HSV),
Tumors varicella, and herpes zoster is easy and immediate in most
cases. Difficulties or doubts may arise in peculiar clinical
Red fluorescence can occur in some malignant skin situations, such as recurrent intraoral or genital HSV (often
tumors, especially squamous cell carcinoma. Conversion of confused with herpetiform or genital aphthous ulcers),
5-aminolevulinic acid to protoporphyrin IX occurs within Kaposi varicelliform eruption (sometimes misdiagnosed as
tumors as the first step in photodynamic therapy and can be impetiginized eczema), atypical chickenpox, and dissemi-
detected with Wood's light.3 nated herpes zoster. In all of these conditions, the Tzanck test
provides a rapid and reliable diagnosis of infection by the
herpes virus group. The cytologic picture is pathognomonic
because of the presence of multinucleated giant keratino-
Tzanck smear cytes, with hyperbasophilic cytoplasm and nuclei exhibiting
a blurry chromatin network (ballooning cells).5,6,8,9
Cutaneous cytodiagnosis was introduced by Arnault The clinical diagnosis of molluscum contagiosum in typical
Tzanck in 1947 as a diagnostic aid for bullous and vesicular sites, and especially in children, is usually straightforward;
dermatoses (pemphigus, herpetic infections) and certain skin however, nonumbilicated lesions with unusual localization,
tumors (basal cell and squamous cell carcinomas), in which mainly in adults, may go unrecognized. The Tzanck smear
neoplastic cells tend to exfoliate easier because of their readily reveals the correct diagnosis owing to the presence of
diminished cell cohesion.5-8 Cytologic examination of skin pathognomonic Henderson-Patterson bodies, the largest known
lesions, also known as the Tzanck test or Tzanck smear, is a inclusion bodies (30 to 35 μm), which are virus-transformed
quick, easy, noninvasive or minimally invasive, inexpensive keratinocytes showing as ovoid, deeply basophilic corpuscles.
procedure to perform in selected cases, with little special Staphylococcal scalded skin syndrome (SSSS), a wide-
equipment or training required. In fact, the Tzanck test is very spread superficial skin loss caused by staphylococcal
simple to perform. The smear is taken from a recent lesion by exfoliatin-type toxins, is observed in infants or children,
gently scraping it with a straight scalpel. The material rarely in adults, with internal (often oropharyngeal) staph-
obtained is smeared on to microscope slides, allowed to air ylococcal infections. In some cases, SSSS can be clinically
dry, and routinely stained with May Grünwald-Giemsa stain confused with toxic epidermal necrolysis (TEN), a wide-
for 20 to 25 minutes. The most useful applications of the spread drug-induced skin loss with a completely different
Tzanck smear in dermatologic practice are listed in Table 2. pathophysiology and prognosis. The Tzanck test allows the
clinician to rapidly differentiate SSSS from TEN. In fact,
Table 2 Cytodiagnosis (Tzanck smear) in dermatologic practice
SSSS is characterized histologically by subcorneal cleavage
with a viable appearance of the epidermis, whereas TEN
Infections shows subepidermal splitting with full-thickness necrosis of
• Herpes simplex the epidermis; therefore, a Tzanck smear taken from a fresh
• Varicella, herpes zoster
lesion shows viable normal (or sometimes acantholytic)
• Molluscum contagiosum
keratinocytes without inflammatory cells in SSSS, and
• Staphylococcal scalded skin syndrome
• Leishmaniasis necrotic keratinocytes with inflammatory (lymphocytes)
Immune disorders and dermal (fibroblasts) cells in TEN.6,8,10
• Pemphigus vulgaris The Tzanck test is the most powerful tool for diagnosis in
• Bullous pemphigoid the early stages of leishmaniasis. The causative protozoa are
• Erosive lichen planus readily detected as light blue ellipsoid bodies with an
• Stevens-Johnson syndrome eccentric nucleus and a smaller kinetosoma (carrying a tiny
• Toxic epidermal necrolysis flagellum) at the opposite pole. They are usually grouped, in
Tumors a typical “swarm of bees” fashion, within the wide cytoplasm
• Sebaceous adenoma of large macrophages (Wright cells). In the chronic form of
• Mastocytoma
infection, there may not be any Leishmania organisms in the
• Basal cell carcinoma
lesions (“untenanted” leishmaniasis), which makes cytology
• Squamous cell carcinoma
• Erythroplasia of Queyrat unhelpful for diagnosis in the late stages.5,6,8
• Paget disease
• Histiocytosis X
Genodermatoses Immune disorders
• Hailey-Hailey disease
• Darier disease In the early stages of pemphigus vulgaris (oral pemphi-
gus), the Tzanck test is probably the most useful diagnostic
Diagnostic procedures in dermatology
procedure because an oral biopsy of painful erosions is
uncomfortable for the patient and of little help for histologic
diagnosis in the absence of the roof of the bulla. The
cytologic pattern is characterized by round epithelial cells,
with a hypertrophic, often atypical neoplasticlike nucleus
and a peripherally basophilic cytoplasm (acantholytic or
“mourning-edged” or Tzanck cells; Figure 1A). Cell
adherence is often observed among leukocytes (leukocyte
adherence) that form chains of white blood cells (“strepto-
cytes”; Figure 1B) and around isolated epitheliocytes that
are surrounded by a ring of leukocytes (Sertoli rosette)
(Figure 1C).5,6,8 When the immunofluorescence technique is
used, Tzanck cells show a typical peripheral fluorescence if
they are isolated (Figure 1D) or have a networklike
appearance resembling that seen in histologic preparations
if they are grouped.11
In bullous pemphigoid, erosive lichen planus, or Stevens-
Johnson syndrome clinically mimicking oral pemphigus, the
Tzanck smear only serves to readily rule out the diagnostic
suspicion of pemphigus. In fact, these three conditions all
lack acantholytic cells. Bullous pemphigoid is cytologically
Fig. 1 Cytology of pemphigus vulgaris: (A) acantholytic cells, (B) streptocytes, (C) Sertoli rosette (all May Grünwald-Giemsa stain, original
magnification ×400), and (D) peripheral immunofluorescence of an acantholytic cell (anti-immunoglobulin G fluorescein serum, original
magnification ×1000).
551
characterized by scarcity of epithelial cells and abundance of
leukocytes (eosinophils in prevalence) often forming chains
(“streptocytes”). A rather nonspecific cytologic picture of
leukocytes, fibrin filaments, altered or necrotic epithelio-
cytes, and rare fibroblasts can be seen in Tzanck smears
taken from erosive lichen planus or Stevens-Johnson
syndrome. In the cases of TEN that might somehow evoke
SSSS, the Tzanck test also proved to be useful, as indicated
in the previous “Infections” section.
Tumors
Several benign or malignant skin tumors can be easily
diagnosed with certainty by means of exfoliative cytology.
Trivial sebaceous adenoma, sometimes resembling an early
basal cell carcinoma, can be recognized immediately by Tzanck
smear, which shows groups of mature sebocytes with large
cytoplasmic vacuoles, basal-type germinative cells, and
transitional cells with an only initial sebaceous differentiation.5
Cytodiagnosis of mastocytoma in infants can easily be
made by a Tzanck smear stained by methylene blue. Plenty
552
of mast cells are recognizable because of their metachroma-
tically stained, reddish purple, cytoplasmic granules.5-8
Basal cell carcinoma is the main indication for the Tzanck
smear, due to both the frequency of the tumor itself and
the high degree of diagnostic reliability it offers for this
neoplasm.5-8,12,13 The pattern is absolutely characteristic, with
clusters of atypical basal cells (basalioid or “basalioma” cells),
which may even exhibit retention of peripheral palisading as in
histologic preparations. Basaloid cells are uniform in size,
elongated, with an oval, deeply basophilic nucleus that occupies
four-fifths of the cell and a scant, poorly defined, also strongly
basophilic cytoplasm that may contain coarse melanin granules
(Figure 2). The reliability of the Tzanck test in diagnosing basal
cell carcinoma also has been confirmed by using a rapid stain
(60 to 90 seconds) with undiluted Giemsa alone.12 Cytodiag-
nosis of basal cell carcinoma is particularly convenient in case
of multiple lesions, involvement of cosmetically sensitive sites
(especially in a young person), suspected recurrence, or when
the tumor has to be treated without a diagnostic biopsy being
taken (eg, with cryosurgery).12,13
Other useful indications of cytodiagnosis in cutaneous
oncology include selected cases of:
• squamous cell carcinoma, especially on mucosal sites
(oral cavity, genitalia), where isolated atypical squa-
mous cells with dysplastic nuclei and irregularly
stained cytoplasm are often diagnostic;
• erythroplasia of Queyrat, characterized by poikilokar-
yosis (ie, nuclear polymorphism relating to size, shape
and staining characteristics), naked, and clumped nuclei;
• Paget disease, with Paget cells highlighted by special
stains, including mucicarmine and periodic acid-
Schiff; and
• histiocytosis X with a peculiar reference to some forms
of Letterer-Siwe disease clinically resembling a trivial
intertrigo but cytologically recognizable for the
presence of atypical Langerhans cells with microva-
cuolated or granular cytoplasm, and large, convoluted,
reniform, indented nucleus.
Fig. 2 Cytology of basal cell carcinoma: basaloid cells contain-
ing coarse melanin granules with peripheral palisading (May
Grünwald-Giemsa stain, original magnification ×400).
E. Ruocco et al.
In all of these conditions, cytodiagnosis is not intended
to be a substitute for analysis of a biopsy specimen,
but can only guide the subsequent diagnostic and thera-
peutic procedures.7
Genodermatoses
Hailey-Hailey disease may present with clinical pictures
resembling eczema, flexural psoriasis, or intertrigo. The
Tzanck test is diagnostic, the smear being characterized by
numerous acantholytic cells with a deeply stained nucleo-
lated nucleus.
In Darier disease, often confused with keratosis pilaris,
the Tzanck test can reveal the typical “mantle cells”
and “corps ronds,” which are better detected in histo-
logic preparations.5,6,8
Microscopic examination for pathogens
Potassium hydroxide
Fungal infections are common skin diseases affecting
millions of people worldwide.14 These infections occur in
both healthy and immunocompromised patients. The
etiologic agents consist of dermatophytes, yeasts, and
nondermatophyte molds. When the diagnosis is not
clinically obvious, laboratory confirmation of fungal infec-
tion, before antifungal treatment is initiated, is accepted as
the gold standard in clinical practice because the antifungal
agents have potential serious side effects and they are
expensive. Therefore, direct microscopic examination is a
highly efficient screening technique15 and is essential
because it allows the clinician to start treatment pending
further investigations.
Microscopic examination requires very thin scales or nail
fragments. The use of thin samples prevents the presence of
air bubbles that could interfere with examination and
therefore facilitates the investigation. A correct visualization
of the fungal elements requires the dissociation of collected
material. The specimens are therefore submitted to clearing
reagents that allow digestion of keratin. Among these
reagents, 10% to 20% potassium hydroxide (KOH), with
or without dimethylsulfoxide, is the most commonly used.16
This method, which is one of the simplest and cheapest,
allows an immediate observation and is particularly suitable
for nails. Because keratin is rapidly digested by KOH, an
immediate examination is required. This limitation may be
overcome by the use of Amann chloral lactophenol, which
allows clearing without heating.
Scales, pustules, blister roofs, and clippings taken from
hair and nails may be examined with this technique. The
collected specimens are transferred onto a microscope slide.
One drop of KOH solution is placed over the material on the
slide. A coverslip is added. The slide is examined with the
Diagnostic procedures in dermatology
condenser is turned down to its lowest position, and the
intensity of the light source is reduced. These two maneuvers
increase the contrast between the fungal hyphae and the
underlying epithelial cells. Superficial fungal diseases
(Figure 3A) of the skin, hair, and nails are commonly
diagnosed using the KOH exam.
This method can also provide a diagnosis in suspected
cases of crusted scabies (Figure 3B), which is becoming
more relevant due to the increase of immunocompromised
patients. A KOH smear from the affected areas is a quick,
simple, and reliable diagnostic tool to identify the adult mites
and their ova. It is easy to perform, requires neither
sophisticated tools nor special skill, and can be done in
less than 5 minutes by every practitioner.17 KOH, however,
usually dissolves the mite fecal material, which is crucial for
diagnosis in the absence of mites or ova. Therefore, when
dealing with atypical cases of scabies, it is preferable to make
use of simple saline, with which numerous fecal pellets can
be detected within seconds.18
Artifacts are commonly present in KOH preparations and
may be confused with hyphae. Cotton threads are thicker and
do not shown parallel sides or branching. Hair has parallel
sides but lacks branching. The edges of epithelial cells
sometimes overlap and appear to form a continuous,
branching line. In such a situation, compression of the
coverslip against the slide induces cells to change position
and separate, thus breaking up the misleading hyphaelike
appearance of the cell outlines.
Visualization of fungal elements at direct examination is
sometimes difficult, particularly for nail specimens and for
inexperienced staff. Staining can increase sensitivity of direct
examination by facilitating the visualization of fungal
structures. The detection of fungal hyphae and spores is
greatly facilitated by the use of fluorochromes such as
calcofluor white. This derivative is a polymer of N-acetyl-D-
glucosamine, which is one of the main polysaccharides of the
fungal cell wall. Calcofluor white can be used in KOH 20%
and allows a rapid and accurate diagnosis of dermatomy-
cosis. Fungal elements in calcofluor-stained specimens
Fig. 3 Potassium hydroxide smear of a skin scraping shows (A) dermatophyte (original magnification ×400) and (B) Sarcoptes scabiei
(original magnification ×20).
553
appear blue when using a fluorescence microscope equipped
with a 330- to 380-nm excitation filter and an emission filter
of 420 nm.19 The only limitation of this method is that it
requires a fluorescence microscope.
Gram stain
Although in recent decades revolutionary advances have
been made in the field of diagnostic procedures, Gram
staining remains a rapid and inexpensive technique for the
diagnostic evaluation of infectious diseases. Gram stain is
used to classify bacteria on the basis of their forms, sizes, and
cellular morphologies and is a critical test for the rapid
presumptive diagnosis of infectious agents (Figure 4). This
simple staining, devised by Gram,20 a Danish pathologist, in
1884 and applied by Ernst21 in 1896 for the staining of
human epidermis, hair, nails and horny organs of other
species, remains a useful test to be performed in every
clinical microbiology laboratory.22
Various modifications of Gram's original procedure
have been proposed over the years. Results from a Gram
stain can confirm the suspicion of an infection within 15
minutes, whereas many other microbiology tests require 24
hours or more. The findings from a Gram stain can be
equivocal and should be assessed carefully in the light of
clinical diagnosis.
The conventional method for performing a Gram stain
begins with a thin, air-dried, heat-fixed preparation on a glass
slide that is flooded with crystal violet and allowed to sit for
at least 30 seconds. The slide is rinsed gently under running
tap water and flooded with Gram's iodine for an additional
30 seconds. After a second tap water rinse, the preparation is
decolorized by rinsing the slide with an acetone-alcohol
solution until dye has been washed out. Finally, the slide
is counterstained for 30 seconds with safranin, rinsed, and
air-dried.23,24
Most bacteria, as well as many fungi and some parasites,
are stained by this method. White blood cells appear
uniformly red, and squamous epithelial cells exhibit a
554
Fig. 4 (A) Gram stain of nodule aspirate shows beaded Gram-positive organisms. (B) Gram-positive yeasts from a cutaneous pustule
(original magnification ×1000).
characteristic mixture of purple and red staining. Gram stain
of skin and tissue specimens is used to diagnose and identify
a variety of infectious diseases. For example, a tissue
fragment from necrotizing fasciitis containing numerous
Gram-positive cocci in chains suggests Streptococcus
pyogenes infection.25,26 In suspected meningococcal disease
Gram staining and culture of hemorrhagic skin lesions are
useful diagnostic tools, especially in patients with sepsis.27,28
The results from the Gram stain may accelerate the diagnosis
and increase the possibility of promptly administering
appropriate therapy.29 In conclusion, it seems appropriate
to take advantage of this old, but rapid and inexpensive
technology, which still helps us improve the diagnosis of
life-threatening diseases.
Molecular biology methods for pathogens
Microscopic identification of fungal elements or bacteria
directly from the clinical specimen is a rapid diagnostic method
but lacks specificity and sensitivity, with false-negative results
in up to 15% of cases. In vitro culture is a specific diagnostic
test, but the technique is too slow to give the results.30,31
Molecular methods, even though they are expensive and
require an experienced staff, are useful when the identification
of a strain is not possible by conventional methods.
The clinical use of antibacterial drugs, immunosuppres-
sive agents after organ transplantation, cancer chemotherapy,
and advances in surgery are associated with an increasing
risk of fungal infections that remain a significant cause of
death in immunocompromised patients or in patients
undergoing invasive procedures. Rapid identification of
yeast isolates from clinical samples is particularly important
given the innately variable susceptibility of yeasts toward
antifungal drugs but is complicated by the increasing number
of emerging pathogenic species.32 Many authors have
demonstrated that gene-specific polymerase chain reaction
(PCR) is highly advantageous as a diagnostic tool for the
E. Ruocco et al.
detection and identification of dermatophytes on direct
application to skin scales or nail specimens.33 Most of these
molecular approaches are used only in research laboratories
for phylogenetic analysis of dermatophytes. These methods
include restriction fragment length polymorphism analysis,34
sequencing of the large submit ribosomal RNA gene35 or the
chitin synthase-encoding, 36 PCR fingerprinting, 37 and
sequencing of the internal transcribed spacer regions.38
Yeasts such as Candida39 and Malassezia40 can also be
identified through molecular methods, which may be applied
in epidemiologic surveys and routine practice. The applica-
tion of a simple PCR using the specific primers provides a
sensitive and rapid identification system for Malassezia spp
from patient skin scales.
Skin lesions are prominent features of many viral
diseases. In some instances, characteristic skin lesions
suggest a specific viral illness, and the diagnosis can be
quickly established. Sometimes other viral diseases may
exhibit similar symptoms. Measleslike lesions can be caused
by HSV type 1 (HSV-1) and HSV type 2 (HSV-2).
Differentiation of suspected cases of measles with molecular
epidemiologic techniques in the laboratory is useful for
measles surveillance. The random PCR screening system41 is
an efficient procedure for the identification of unknown viral
pathogens and may be useful in the differential diagnoses of
measleslike diseases. Rapid and reliable detection of
varicella zoster virus (VZV), HSV-1, and HSV-2 is of
clinical significance in immunocompromised patients and
patients with infections of the central nervous system.
Because antiviral drugs are available and rapidly
effective, a correct laboratory diagnosis of these viral
infections is crucial. PCR or real-time PCR42,43 has the
advantage of rapid amplification, a reduced risk for
contamination, and is a suitable method for the diagnosis
of VZV and HSV in specimens from skin lesions. In
addition, a combination of type-specific and degenerate PCR
methods can be used to achieve DNA detection of human
papillomaviruses, which were alleged to play a pathogenic
role in psoriasis.44
Diagnostic procedures in dermatology
Tuberculosis continues to be a serious medical problem
today. Cutaneous tuberculosis shows considerable morpho-
logic variability and is included in the differential diagnosis
of many other skin disorders. Reliable laboratory tests are
needed to confirm or rule out this diagnosis. 45 The
identification of Mycobacterium tuberculosis and related
organisms remains difficult by conventional diagnostic tools
such as microscopy and culture. PCR has emerged as a
promising tool in the diagnosis of various forms of cutaneous
tuberculosis and atypical mycobacteria infections, although a
careful clinical examination is still the gold standard for the
evaluation and treatment of cutaneous tuberculosis and
atypical mycobacteria skin infections.46
Modern molecular technologies have a substantial affect
in many areas of parasitology. Cutaneous and mucocutane-
ous leishmaniasis present similar clinical appearances but
have a different prognosis. Another problem is distinguish-
ing between true Leishmania infection and apparently similar
skin diseases in endemic areas. Conventional methods for the
diagnosis have low sensitivities and are unable to quantify
the number of viable parasites.47,48 This represents a major
obstacle for the diagnosis of the disease and for the study of
the effectiveness of treatments. PCR tests (quantitative
nucleic acid sequence-based amplification, real-time reverse
transcriptase PCR, and real-time PCR) applied to biopsy
specimens proved useful for the differential diagnosis of
cutaneous leishmaniasis and may provide an important tool
for monitoring the therapy.49
Conclusions
The introduction of molecular biology methods for the
detection and identification of pathogens has improved the
speed and accuracy of laboratory diagnosis in recent years.
These methods are intended as a complement to, not as a
replacement for, the already applied microbiologic methods.
The integrated analysis of all of them is bringing the most
efficient results.
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