Abstract
-
A diagnosis, in the sense of diagnostic procedure, can be regarded as an attempt at classification of an individual’s condition into separate and distinct categories that allow medical decisions about treatment and prognosis to be made. A diagnostic test is any kind of medical test performed to aid in the diagnosis or detection of disease and to provide prognostic information on people with established disease.
-
History taking is of paramount importance in assessing infectious disease. By careful and systematic questioning, it is possible to assess the factors pertinent to differential diagnosis and particular lines for further investigation.
-
The first look is best made without prejudices of former diagnoses and without bias of laboratory data. In many instances, a specific diagnosis is made in a fraction of a second if it is a simple matter of recognition. The informed look is the one most practiced by dermatologists; it comes from knowledge, experience, and visual memory. Where the diagnosis doesn’t come from a glance, the diagnostic tests come in.
-
Probabilistic reasoning is the specific use of symptoms, signs, or diagnostic tests to rule in or rule out a diagnosis. Probabilistic reasoning requires knowing the degree to which a positive or negative result of a test adjusts the probability of a given disease.
-
Some experts suggest that dermoscopy improves diagnostic capability beyond simple clinical inspection. Therefore, dermoscopy is gaining popularity in daily clinic practice as a valuable tool in differential diagnosis of hair and scalp disorders. However, as a diagnostic procedure, dermoscopy remains to be understood as representing an integral part of a more comprehensive dermatological examination. It would be unwise to choose shortcuts and replace time-tested examination techniques with a higher sensitivity and specificity by dermoscopy.
-
Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye. There are three well-known branches of microscopy: optical, electron, and scanning probe microscopy, including the trichogram, hair shaft analysis, and histopathologic examination.
-
Microbiologic studies are mandatory in inflammatory conditions of the scalp with scaling, crusting, and/or pustulation. All clinical providers are recommended to ascertain the causative organism in any infectious disease, either through direct visualization, culture, polymerase chain reaction assay, or blood tests where applicable.
-
Imaging provides an important adjunct to the physical examination, allowing evaluation for lymphadenopathy in regions that are not externally accessible, such as the mediastinum, or intra-abdominal sites, assessment of internal organs for evidence of infection, particularly the lungs, the liver, the kidneys, and the brain.
-
Ultimately, the dermatologist participates with the other medical disciplines in the diagnosis and treatment of infections as they may relate to systemic disease.
Similar content being viewed by others
Diagnosis is the identification of the nature and cause of a certain phenomenon. Diagnosis is used in many different disciplines, with variations in the use of logic, analytics, and experience, to determine cause and effect.
Medical diagnosis is the process of determining which disease or condition explains a person’s symptoms and signs. It is most often referred to as diagnosis with the medical context being implicit. The information required for diagnosis is typically collected from a history and physical examination of the person seeking medical care. Often, one or more diagnostic procedures, such as medical tests, are also done during the process.
Diagnosis is often challenging, because many signs and symptoms are nonspecific. For example, erythema, by itself, is a sign of many disorders and thus does not tell the healthcare professional what is wrong. Therefore, differential diagnosis in which several possible explanations are compared and contrasted must be performed. This involves the correlation of various pieces of information followed by the recognition and differentiation of patterns. In a pattern recognition method, the provider uses experience to recognize a pattern of clinical characteristics. It is mainly based on certain symptoms or signs being associated with certain diseases or conditions, not necessarily involving the more cognitive processing involved in a differential diagnosis. Occasionally the process is made easy by a sign or symptom or a group of several that are pathognomonic.
A diagnosis, in the sense of diagnostic procedure, can be regarded as an attempt at classification of an individual’s condition into separate and distinct categories that allow medical decisions about treatment and prognosis to be made.
A diagnostic test is any kind of medical test performed to aid in the diagnosis or detection of disease and to provide prognostic information on people with established disease.
The term “diagnostic criteria” designates the specific combination of signs and symptoms and test results that the clinician uses to attempt to determine the correct diagnosis.
A medical algorithm is any computation, formula, statistical survey, nomogram, or look-up table, useful in healthcare. Medical algorithms include decision tree approaches to healthcare treatment and also less clear-cut tools aimed at reducing or defining uncertainty. The intended purpose of medical algorithms is to improve and standardize decisions made in the delivery of medical care. Medical algorithms assist in standardizing selection and application of treatment regimens. However, algorithms are based on a typical patient with a typical condition. Clinical algorithms may be useful for the average diagnosis and treatment, but they fail when a doctor needs to think outside of their boxes, when symptoms are vague, multiple, or confusing, and when test results are inconclusive. Doctors who turn down their own thinking on the authority of classification schemes and algorithms have a statistic way of looking at people. But statistics embody averages, not individuals. Ultimately, computations obtained from medical algorithms should be compared with, and tempered by, clinical knowledge and physician judgment.
For a diagnostic workup in infectious diseases, access to the following diagnostic tools and facilities are required:
-
Patient history (hair specific, general, psychosocial)
-
Clinical examination (scalp, complete skin, nails, mucous membranes)
-
Dermoscopy
-
Microscopic examination (light, polarization, and scanning electron microscopy)
-
Hair pluck (trichogram)
-
Scalp biopsy
-
Histopathology, including immunofluorescence
-
Wood lamp examination
-
Mycology, including KOH preparation and fungal cultures
-
Other microbiological services, including PCR
-
Blood test facilities (phlebotomy and laboratory services: CRP, CBC, VDRL, TPHA, Quantiferon-Tbc test, others)
-
Imaging
-
Access to non-dermatological clinical disciplines
11.1 Patient History
History taking is of paramount importance in assessing infectious disease. By careful and systematic questioning, it is possible to assess the factors pertinent to differential diagnosis and particular lines for further investigation.
In the course of history taking, it is advisable never to accept anything for true, neither from the patient nor from the referring physician, which is not clearly recognizable as such, that is to say, carefully to avoid precipitancy and prejudice, and to comprise nothing more in one’s judgment than what is presented to the mind so clearly and distinctly as to exclude all ground of doubt.
William Osler (Canadian physician, 1849–1919) said “If you listen to the patient, he is telling you the diagnosis.” How a doctor asks questions is key to patient activation and engagement. The way a doctor poses his questions, structures the patient’s answers, and at the same time gives the patient the feeling that the doctor is really interested in hearing what he has to say.
The six components of a patient history are:
-
Chief concern
-
History of present illness
-
Past medical history
-
Family history
-
Sociocultural history
-
Review of systems
Specific components relevant to infectious diseases require particular attention. In general, these aspects focus on two areas:
-
1.
An exposure history that may identify microorganisms with which the patient may have come into contact
-
2.
Host-specific factors that may predispose to the development of an infection
Although the social history taken by physicians is often limited to inquiries about a patient’s alcohol and tobacco use, a complete social history can offer a number of clues to the underlying diagnosis. Knowing whether the patient has any high-risk behaviors, such as unsafe sexual behaviors, and IV drug use, potential hobby-associated exposures, such as avid gardening with possible Sporothrix schenckii exposure, animal exposure, such as increased risk for Microsporum canis or Trichophyton mentagrophytes infection, and tick-borne diseases, or occupational exposures, such as increased risk for Mycobacterium tuberculosis exposure in funeral service workers, can facilitate diagnosis. Finally, attention should be paid to both international and domestic travels. Domestic travel may have exposed patients to pathogens that are not normally found in their local environment and therefore may not routinely be considered in the differential diagnosis. Beyond simply identifying locations that a patient may have visited, the physician needs to delve deeper to learn what kinds of activities and behaviors the patient engaged in during travel, such as the types of food and sources of water consumed, freshwater swimming, animal exposures, and whether the patient had the necessary immunizations and/or took the necessary prophylactic medications prior to travel.
Because many opportunistic infections affect only immunocompromised patients, it is of vital importance to determine the immune status of the patient. Defects in the immune system may be due to malnutrition, an underlying disease, such as malignancy and HIV infection; a medication, particularly glucocorticosteroids and immunosuppressives; a treatment modality, such as splenectomy, chemotherapy, or total body irradiation; or a primary immunodeficiency condition.
Many infections are caused by members of the indigenous microbiota. These infections typically occur when these microbes escape their normal habitat and enter a new one. Thus, maintenance of epithelial barriers is one of the most important mechanisms in protection against infection. However, hospitalization of patients is often associated with breaches of these barriers, such as placement of IV lines, surgical drains, or tubes that allow microorganisms to localize in sites to which they normally would not have access. Accordingly, knowing what lines, tubes, and drains are in place is helpful in ascertaining what body sites might be infected.
Finally, knowledge about a patient’s previous infections, with the associated microbial susceptibility profiles, is very helpful in determining possible etiologic agents. Specifically, knowing whether a patient has a history of infection with drug-resistant organisms, such as methicillin-resistant Staphylococcus aureus, or may have been exposed to drug-resistant microbes during a recent stay in a hospital, nursing home, or long-term acute-care facility may alter the choice of empirical antibiotics.
11.2 Clinical Examination
The skin and hair are gratifying for diagnosis. One has but to look, and recognize, since everything to be named is in full view. Looking would seem to be the simplest of diagnostic skills, and yet its simplicity lures one into neglect. To reach the level of artistry, looking must be a skillful active undertaking. The skill comes in making sense out of what is seen, and it comes in the quest for the underlying cause, once the disorder has been named. The first look is best made without prejudices of former diagnoses and without bias of laboratory data. In many instances a specific diagnosis is made in a fraction of a second if it is a simple matter of recognition. The informed look is the one most practiced by dermatologists; it comes from knowledge, experience, and visual memory. Where the diagnosis doesn’t come from a glance, the diagnostic tests come in, i.e., the dermatological techniques of examination, and the laboratory evaluation.
The fact that many infections have cutaneous manifestations gives the skin examination particular importance in the evaluation of patients. Pattern recognition relies both on the specificity of a particular disease pattern and on the diagnostic skill of the physician. It relies on symptoms and signs compared to previous patterns or cases and on the memory of known patterns.
Spot diagnosis arises from an unconscious recognition of a particular nonverbal pattern, usually visual. The spot diagnosis is almost instantaneous, relies on previous nonverbal experience of the condition, and does not require further history from the patient to trigger the possible diagnosis. Many consider a spot diagnosis as basically pattern recognition. The main determinant in the use of spot diagnosis is clinical experience with a given condition.
Red flags are specific symptoms or signs that may be volunteered by the patient or may need to be elicited in the history or examination to rule out a serious condition, for example, checking for neck stiffness in a patient with headache to rule out meningitis. If the symptom or sign cannot be ruled out, it triggers action, which can range from a more detailed physical examination to hospital referral.
Probabilistic reasoning is the specific use of symptoms, signs, or diagnostic tests to rule in or rule out a diagnosis. Probabilistic reasoning requires knowing the degree to which a positive or negative result of a test adjusts the probability of a given disease [1].
11.3 Dermoscopy
The naked eye is right for the global look, but for close inspection, the additional use of a magnifying glass is practiced. The handheld, single-lens magnifier is the simplest and least expensive, most commonly used by dermatologists, usually at a magnification of 3× to 4×. Although the pathologist lives in a world magnified 100 to 1000 times, the clinician doesn’t benefit from a highly magnified view of the patient, lest he performs dermoscopy (10×) and is knowledgeable of the clinicopathologic correlations.
Dermoscopy is a noninvasive diagnostic tool that permits recognition of morphologic structures not visible to the naked eye. Dermatologists involved in the management of and scalp disorders have discovered dermoscopy to also be useful in their daily clinical practice. Scalp dermoscopy is not only helpful for the diagnosis of hair and scalp disorders, but it can also give clues about the disease stage and progression.
Some experts suggest that the use of dermoscopy in the clinical evaluation of hair and scalp disorders improves diagnostic capability beyond simple clinical inspection and reveals novel features of disease, which may extend our clinical and pathogenetic understanding. Therefore, dermoscopy of hair and scalp is gaining popularity in daily clinic practice as a valuable tool in differential diagnosis of hair and scalp disorders. This method allows viewing of the hair and scalp at high magnifications using a simple handheld dermatoscope (Heine Delta 20®, DermoGenius®, DermLite II PRO HR®, or DermLite DL3® (Fig. 11.1a) can be used, with alcohol as the interface solution.
(a–g) Dermoscopy: (a) different models of hand held dermoscopes; (b) dermoscopy of tinea capitis, comma hairs and corkscrew hairs; (c) of syphilitic alopecia, reduction in the number of terminal hairs, (d, e) of pediculosis capitis, (d) the louse (e) and nits; (f) of a wart; and of (g) folliculitis decalvans, hair tufting, follicular pustule, and capillary loops
Using dermoscopy, signature patterns are seen in a range of scalp and hair conditions. Some predominate in certain diseases; others can even help make a diagnosis in clinically uncertain cases.
Dermoscopic findings in specific infectious diseases of the hair and scalp have been comma hairs and corkscrew hairs in tinea capitis [2] (Fig. 11.1b). Dermoscopy has proven useful to identify parasitism of hair of the beard in tinea barbae, just as it has proven useful in the diagnosis of tinea capitis [3].
Dermoscopy of the moth-eaten areas in syphilitic alopecia showed that alopecia is mainly due to a reduction in the number of terminal hairs [4] (Fig. 11.1c).
In pediculosis capitis, either the lice (Fig. 11.1d) or nits (Fig. 11.1e) are easily visualized through the dermoscope, and empty cases can reliably be differentiated from nymph-containing viable eggs [5].
In children, important dermoscopic structures seen in infectious and inflammatory skin conditions and hair disorders, such as scabies, pediculosis, phthiriasis, molluscum contagiosum, tinea nigra, and verrucae are well-characterized dermoscopically by delta-shaped structures, ovoid-shaped nits, the crab louse, red corona, brown strands or spicules, and multiple densely packed papilla with a central black dot surrounded by a whitish halo (Fig. 11.1f), respectively [6].
Dermoscopy may support the recognition of folliculitis etiology. In an observational study on 240 patients with folliculitis determined on the basis of clinical and dermoscopic assessments, dermoscopic images of the most representative lesions were acquired for each patient, and etiology was determined on the basis of cytologic examination, culture, and histologic examination. Dermoscopic images were evaluated according to predefined diagnostic criteria by a dermatologist who was blinded to the clinical findings. Of the 240 folliculitis lesions examined, 90% were of infectious and 10% of noninfectious origin. Infectious folliculitis was caused by parasites (n = 71), fungi (n = 81), bacteria (n = 57), or seven viruses (n = 7). The overall accuracy of dermoscopy was 73.7%. Dermoscopy showed good diagnostic accuracy for Demodex (88.1%), scabietic (89.7%), and dermatophytic folliculitis (100%), as well as for pseudofolliculitis (92.8%) [7].
Dermoscopic features of folliculitis decalvans are severe scaling and crusting, pronounced hair tufting, follicular pustules, and numerous coiled capillary loops (Fig. 11.1g) [8]. Based on dermoscopic findings, ultimately a trichoscopy activity scale for folliculitis decalvans has been proposed, though short of correlations with histopathological or microbiological studies [9].
In summary, as a diagnostic procedure, dermoscopy remains to be understood as representing an integral part of a more comprehensive dermatological examination. Moreover, dermoscopy of the hair and scalp also represents an integral part of surface or epiluminescence microscopy of the skin, given that an important portion of respective signature patterns relates to the condition of the scalp skin rather than to the hair, and as such should retain its original designation as dermoscopy versus the sectarian term trichoscopy. Finally, it would be unwise to choose shortcuts and replace time-tested examination techniques with a higher sensitivity and specificity with dermoscopy, specifically the hair pluck in telogen effluvium, the light microscopic hair shaft analysis in the disorders of the hair shaft, the microbiological studies in the pustulofollicular and granulomatous diseases of the scalp, the scalp biopsy for histopathological examination, histochemical and microbiological studies in the scarring alopecias, and serologic testing in syphilis, HIV, and other systemic infectious diseases [10].
11.4 Microscopic Examination
Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye. There are three well-known branches of microscopy: optical, electron, and scanning probe microscopy.
Optical or light microscopy involves passing visible light transmitted through or reflected from the sample through a single lens or multiple lenses to allow a magnified view of the sample. The resulting image can be detected directly by the eye, imaged on a photographic plate, or captured digitally. The single lens with its attachments, or the system of lenses and imaging equipment, along with the appropriate lighting equipment, sample stage, and support, makes up the basic light microscope.
Live cells in particular generally lack sufficient contrast to be studied successfully, since the internal structures of the cell are colorless and transparent. The most common way to increase contrast is to stain the structures with selective dyes, but this involves fixing the sample. Staining may introduce artifacts, which are apparent structural details that are caused by the processing of the specimen and are thus not features of the specimen (FDS).
To improve specimen contrast or highlight structures in a sample, special techniques must be used:
Bright-field microscopy is the simplest of all the light microscopy techniques. Sample illumination is via transmitted white light illuminated from below and observed from above. Limitations include low contrast of most biological samples and low apparent resolution due to the blur of out-of-focus material. The simplicity of the technique and the minimal sample preparation required are significant advantages.
Dark-field microscopy is a technique for improving the contrast of unstained, transparent specimens. Dark-field illumination uses a carefully aligned light source to minimize the quantity of directly transmitted unscattered light entering the image plane, collecting only the light scattered by the sample. Dark field can dramatically improve image contrast, particularly of transparent objects, while requiring little equipment setup or sample preparation. However, the technique suffers from low light intensity in the final image of many biological samples and continues to be affected by low apparent resolution. Since 1909, examinations of unstained and unfixed preparations by dark-field microscopy have been able to show with complete certainty the presence of infection caused by spirochetes, even before the appearance of the serologic tests. Dark-field microscopy allows visualization of live treponemes obtained from a variety of cutaneous or mucous membrane lesions. In primary syphilis, the chancre teems with treponemes that can be seen with dark-field microscopy. A positive dark-field result is an almost certain diagnosis of primary, secondary, or early congenital syphilis. Of note, the mouth harbors normal nonpathogenic treponemes that are indistinguishable microscopically from Treponema pallidum. Therefore, oral specimens cannot be used for dark-field microscopy because of the possibility of false-positive test results.
Polarized light microscopy can mean any of a number of optical microscopy techniques involving polarized light. Simple techniques include illumination of the sample with polarized light. Directly transmitted light can, optionally, be blocked with a polarizer orientated at 90 degrees to the illumination. More complex microscopy techniques which take advantage of polarized light include differential interference contrast microscopy and interference reflection microscopy. Scientists will often use a device called a polarizing plate to convert natural light into polarized light. These illumination techniques are most commonly used on birefringent samples where the polarized light interacts strongly with the sample and so generating contrast with the background. Polarized light microscopy is used extensively in optical mineralogy. In pathology, Schaumann bodies are birefringent calcium and protein inclusions inside of Langhans giant cells as part of a granuloma, typically seen in sarcoidosis and less commonly in tuberculosis.
Fluorescence microscopy. When certain compounds are illuminated with high energy light, they emit light of a lower frequency. This effect is known as fluorescence. Often specimens show their characteristic autofluorescence image, based on their chemical makeup. This method is of critical importance in the modern life sciences, as it can be extremely sensitive, allowing the detection of single molecules. Many fluorescent dyes can be used to stain structures or chemical compounds. One powerful method is the combination of antibodies coupled to a fluorophore as in immunostaining. Examples of commonly used fluorophores are fluorescein or rhodamine. The antibodies can be tailor-made for a chemical compound.
Calcofluor White (CFW) has become a valuable and routine reagent in the clinical mycology laboratory. It binds to β-1–3 and 1–4 polysaccharides, such as cellulose and chitin present in fungal cell walls, and fluoresced when exposed to long-wave UV light.
Until the invention of sub-diffraction microscopy, the wavelength of the light limited the resolution of traditional microscopy to around 0.2 μm. In order to gain higher resolution, the use of an electron beam with a far smaller wavelength is used in electron microscopes.
Transmission electron microscopy (TEM) is quite similar to the compound light microscope, by sending an electron beam through a very thin slice of the specimen. The resolution limit in 2005 was around 0.05 [dubious-discuss] nanometer and has not increased appreciably since that time.
Scanning electron microscopy (SEM) visualizes details on the surfaces of specimens and gives a very nice 3D view. It gives results much like those of the stereo light microscope. The best resolution for SEM in 2011 was 0.4 nm.
11.5 Trichogram
Many factors can lead to a pathologically increased hair loss, including febrile infections. Whatever the cause, the follicle tends to behave in a similar way.
Telogen effluvium results from late onset increased shedding of hairs from the telogen phase of the hair cycle and represents by far the commonest cause of hair loss. An increase in the percentage of follicles in telogen >20% leads to increased shedding of hairs in telogen. This can either be due to synchronization phenomena of hair cycling, with shedding of hairs in the hundreds in telogen effluvium or due to a decrease of anagen phase duration in androgenetic alopecia with its sex-typical patterns of alopecia, variable shedding of hair, and increasing diversity of hair shaft diameters.
Dystrophic anagen effluvium is an early-onset hair loss that results from the shedding of large numbers of hairs from the anagen phase of growth. It is a major characteristic of anagen that the epithelial hair follicle compartment undergoes proliferation, with the hair matrix keratinocytes showing the highest proliferative activity in building up the hair shaft. The common pathogenesis which unites the different etiologies of dystrophic anagen effluvium is a direct insult to the rapidly dividing bulb matrix cells.
Postfebrile effluvium has traditionally been categorized as telogen effluvium, yet it may present with different pathomechanisms and clinical patterns. Evidence exists that the hair follicle may respond to infection with both shedding patterns, telogen effluvium, or dystrophic anagen effluvium, depending on the type and intensity of the insult.
The trichogram or hair pluck test is a semi-invasive technique for hair analysis on the basis of the hair growth cycle. It involves the forceful plucking of 50–100 hairs with a forceps from specific sites of the scalp and microscopic examination of the hair roots (Fig. 11.2a–o). A major objective of trichogram measurements is to evaluate and count the status of individual hair roots (Fig. 11.3a–e) and to establish the ratio of anagen to telogen roots. Original studies on the dynamics of the follicular cycle have largely depended on the microscopic evaluation of plucked hairs with quantitative measuring of the number of individual hair roots. Subsequently, the trichogram technique was developed and standardized to serve as a diagnostic tool for evaluation of hair loss in daily clinical practice. For this purpose it is simple to perform, repeatable, and reasonably reliable under standardized conditions. The trichogram technique provides reliable results under the condition that hair samples are obtained under a standardized procedure.
(a–o) Trichogram technique. (a) The materials necessary for performing a trichogram include a tail comb, hair clips, artery forceps covered with rubber tube, a pair of scissors, microslides, 76 × 26 mm, cover glasses, 50 × 24 mm, Eukitt, xylol, a dissecting needle, and a binocular microscope with variable objectives (2.5 and 4.0. Usually, epilations are carried out from two specified sites at the same time: in diffuse effluvium or androgenetic alopecia frontal (2 cm behind the forehead and 2 cm lateral) and occipital (2 cm lateral from the occipital protuberance) and in circumscribed alopecias one sample is taken from the border zone and the control from the normal appearing contralateral region. (b–e) Within the chosen area for epilation, the hair is parted and fixed with clips. (f, g) Along the parting line, a bundle of approximately 50 to 100 hairs is lifted parallel along the course of the hair and grasped close to the scalp with the forceps whose jaws are covered with the rubber tubes. (h–j) The forceps jaws are pressed together to the maximum and the tuft of hair is then epilated. A sharp quick pull and exact plucking in the direction of the emergence angle of the hairs form the scalp are important to obtain a reliable hair root pattern. Slow or hesitant traction or the wrong pulling direction may induce distortions or alterations of the plucked hairs complicating interpretation. (k–n) The procedure is repeated at the second site. (o) Embedding of epilated hairs occurs immediately to prevent dehydration of the hair roots. A few drops of Eukitt (after condensation, dilute with xylol) are given on two marked microslides. The tuft of hairs is taken with thumb and pointing finger; the roots are dipped in the embedding material (Eukitt), cut off 2 cm above the roots, and arranged in a fast manner with the dissecting needle in a parallel position before being covered. The evaluation can be done when the embedding material no longer runs, usually after 10 min. Correctly embedded hair roots are suitable for unlimited storage
Since in 95% of cases hair loss is due to a disorder of hair cycling, trichogram measurements serve as a standard method for quantifying the hair in its different growth cycle phases as it relates to the pathologic dynamics underlying the loss of hair. The percentage of hair roots in anagen, catagen, or telogen reflects either synchronization phenomena of the hair cycle or alterations in the duration of the respective growth cycle phases, while the presence of dystrophic hair roots signalizes a massive damage to anagen hair follicles.
To date, no standardization defines the best way to fix the hair shafts for reading under the microscope. A review of 76 articles indexed in PubMed with the keywords “trichogram” and “technique” published from 1970 to 2021 showed that only 14 studies (18.4%) mentioned some liquid or other fixation media when conducting the technique. Of these, one used formaldehyde (7.15%), two used a drop of Canadian balsam (14.28%), three used only a thin glass slide cover (21.42%), two used double-sided tape (14.28%), five used adhesive tape (37.71%), one used unspecified liquid (7.15%), and 62 (81.57%) did not mention or did not use any form of fixation of the hair strands.
A mixture of 45% acrylic resin and 55% xylenes (Eukitt®) can be used for fixing and reading the trichogram with excellent results. It provides little formation of air bubbles and facilitates exam interpretation, especially for inexperienced examiners. On the other hand, this technique is a more expensive option, and it’s more challenging to be found in some parts of Brazil. In turn, the use of liquids that do not promote adherence of the hair strands to the glass slide, such as formaldehyde, 0.9% saline solution, and distilled water, facilitates the movement of hair strands on the slide, making it difficult to visually analyze and count the hairy roots in different optical fields. In the authors’ experience, using a transparent enamel base coat (basically, clear nail polish) is a cheap, easy-to-access, and helpful strategy when preparing the hair shafts for the trichogram. When opting for this strategy, the examiner must place the hair strands on a previously prepared slide with a generous amount of enamel base coat and then cover with a coverslip glass slide (Fig. 11.4a–d). Drying is quick, and fixation is adequate, with minimal air bubbles formation (Fig. 11.5a, b). Also, the material can be kept for analysis on subsequent days [11].
11.6 Histopathology
In some cases of alopecia, a diagnosis cannot be made based on results of physical examination, diagnostic hair techniques, and laboratory studies. This is particularly the case in the scarring alopecias. In these cases, a scalp biopsy for histopathological examination may provide the specific diagnosis.
For a biopsy an area of the scalp is chosen where the disease is active, frequently the margin of the involved area shows the pathologic changes best, while areas should be avoided where there are no hair follicles present. After choosing the appropriate site, the hairs are clipped in a 1 cm2 area, leaving a 2 mm stubble (Fig. 11.6a). The area is prepared with 70% alcohol. For adequate anesthesia and hemostasis, 1.5 ml of 1.0% lidocaine with epinephrine is injected raising a large wheal. To obtain an adequate vasoconstrictor effect, it is advisable to wait 20–30 min before proceeding to the biopsy. Also, areas are to be avoided that lie over the temporal or occipital arteries or in which an arterial palpation can be detected. To avoid tying long hairs in the suture material, paper tape is placed over the uncut hairs surrounding the biopsy site. An adequate biopsy specimen can be obtained by using a 6 mm punch instrument that is placed parallel to the emerging angle of the hair stubbles. The punch is turned through the dermis and subcutaneous fat to a level including the hair bulbs. The biopsy specimen can be grasped at the edge with a fine-toothed forceps, while it is cut free of attachment deep in the fat with a small, curved scissors. Alternatively, a thin 1 cm ellipse can be made, especially if the scalp is very tight or scarred, and a 6 mm punch site may not be able to be closed with sutures. The biopsy site is sutured with blue 4-0 Proline. Three to four stitches are usually adequate for hemostasis.
(a–g) Scalp biopsy. (a) For a biopsy an area of the scalp is chosen where the disease is active, frequently the margin of the involved area shows the pathologic changes best, while areas should be avoided where there are no hair follicles present. After choosing the appropriate site, the hairs are clipped in a 1 cm2 area, leaving a 2 mm stubble. The area is prepared with 70% alcohol. (b) For adequate anesthesia and hemostatis, 1.5 ml of 1.0% lidocaine with epinephrine is injected raising a large wheal. To obtain an adequate vasoconstrictor effect, it is advisable to wait 20–30 min before proceeding to the biopsy. Also, areas are to be avoided that lie over the temporal or occipital arteries or in which an arterial palpation can be detected. To avoid tying long hairs in the suture material, paper tape is placed over the uncut hairs surrounding the biopsy site. (c) An adequate biopsy specimen can be obtained by using a 6 mm punch instrument that is placed parallel to the emerging angle of the hair stubbles. The punch is turned through the dermis and subcutaneous fat to a level including the hair bulbs. (d) The biopsy specimen can be grasped at the edge with a fine-toothed forceps, while it is cut free of attachment deep in the fat with a small, curved scissors. Alternatively, a thin 1 cm ellipse can be made, especially if the scalp is very tight or scarred, and a 6 mm punch site may not be able to be closed with sutures. (e) The biopsy site is sutured with blue 4-0 Proline. Three to four stitches are usually adequate for hemostasis. (f) The specimen is then cut in half with a #15 blade parallel to the longitudinal axis of the hair shafts. (g) One half of the specimen is submitted for the routine hematoxylin and eosin examination, while the other half for immunofluorescence studies as indicated. In some instances, transverse sectioning of a second, entire punch according to the Headington technique may be done for quantitative morphometric analyses of the follicles and hair
Problems related to the scalp biopsy may be the reluctance of dermatologists to perform a scalp biopsy and therefore lack of experience with the proper procedure and the lack of familiarity of some pathologists with scalp histopathology. Nevertheless, if done and examined properly, the scalp biopsy is an easy, relatively painless, and bloodless procedure that represents an invaluable adjunct for confirming or establishing the diagnosis of a specific type of alopecia, whether of infectious or noninfectious origin.
Histopathology refers to the microscopic examination of tissue in order to study the manifestations of disease. Specifically, in clinical medicine, histopathology refers to the examination of a biopsy or surgical specimen by a pathologist, after the specimen has been processed and histological sections have been placed onto glass slides. The tissue is removed from the body and placed after dissection in a fixative. The most common fixative is 10% neutral buffered formalin (corresponding to 3.7% w/v formaldehyde in neutral buffered water, such as phosphate-buffered saline). Water is removed from the sample in successive stages by the use of increasing concentrations of alcohol. Xylene is used in the last dehydration phase instead of alcohol—this is because the wax used in the next stage is soluble in xylene where it is not in alcohol allowing wax to permeate the specimen. The wax-infiltrated specimen is then transferred to an individual specimen embedding container. Finally, molten wax is introduced around the specimen in the container and cooled to solidification so as to embed it in the wax block. This process is needed to provide a properly oriented sample sturdy enough for obtaining a thin microtome section for the slide. Once the wax-embedded block is finished, sections will be cut from it and usually placed to float on a water bath surface which spreads the section out. A number of slides will usually be prepared from different levels throughout the block. After this the thin section mounted slide is stained, and a protective coverslip is mounted on it. To see the tissue under a microscope, the sections are stained with one or more pigments. The aim of staining is to reveal cellular components; counterstains are used to provide contrast. For common stains, an automatic process is normally used.
The most commonly used stain in histology is a combination of hematoxylin and eosin (H&E). Hematoxylin is used to stain nuclei blue, while eosin stains the cytoplasm and the extracellular connective tissue matrix of most cells pink. There are hundreds of various other techniques which have been used to selectively stain cells. Other compounds used to color tissue sections include gram stain, Brown-Brenn modified gram stain, periodic acid shift (PAS), Giemsa, and silver salts for visualization of pathogens. The histological slides are examined under a microscope by a pathologist, and the medical diagnosis is formulated as a pathology report describing the histological findings and the opinion of the pathologist as it refers to the request of the clinician. For this purpose, effective communication between the clinician and pathologist is important.
11.7 Wood Light Examination
Wood light examination is an examination technique used in dermatology by which ultraviolet light is shone at a wavelength of approximately 365 nm onto the skin of the patient, for observation of any subsequent fluorescence. A black light, also called an ultraviolet (UV)-A light, or Wood’s lamp is a lamp that emits long-wave ultraviolet light (UV-A) and very little visible light (Fig. 11.7a, b). One type of lamp has a violet filter material, either on the bulb or in a separate glass filter in the lamp housing, which blocks most visible light and allows through UV, so the lamp has a dim violet glow when operating. Ultraviolet radiation is invisible to the human eye, but illuminating certain materials with UV radiation causes the emission of visible light, causing these substances to glow with various colors. This is called fluorescence and has many practical uses. Black lights are essential when UV-A light without visible light is needed, particularly in observing fluorescence. Black lights, for instance, are a common tool for rock-hunting and identification of minerals by their fluorescence (Fig. 11.8: collection of minerals fluorescing under a black light).
In medicine, such a light source is referred to as a Wood’s lamp, named after American physicist Robert Williams Wood (1868–1955), who invented the original Wood’s glass UV filters. Though the technique for producing a source of ultraviolet light was devised by Robert Williams Wood in 1903, it was only in 1925 that the technique was used introduced into dermatology for the detection of fungal infection of hair. The technique has many uses, both in distinguishing fluorescent conditions and in locating the precise boundaries of the condition. Specifically, it is helpful in diagnosing particular fungal and bacterial infection. The technique is performed in the dark. Normal healthy skin is slightly blue but shows white spots where there is thickened skin, yellow where it is oily, and purple spots where it is dehydrated.
Fluorescence of Microsporum canis infections is apple green, Corynebacterium minutissimum coral red (Fig. 11.9), Pseudomonas yellow-green, and Cutibacterium acnes an orange glow.
More recently, band-like green fluorescence of nails and hair on Wood’s lamp examination has been observed in the course of the COVID-19 pandemic and been attributed to the use of high-dose favipiravir [12]. The drug’s active phosphorylated metabolite was shown in human plasma, and its concentration and fluorescence intensity had shown a linear relationship [13].
Favipiravir is a broad-spectrum antiviral, effective against RNA viruses including influenza and Ebola, and one of the current medications approved for the treatment of COVID-19. The drug selectively inhibits the RNA-dependent RNA polymerase by acting as a guanine analog. It transforms into an active phosphoribosylated form in cells, causing chain termination, slowing viral RNA synthesis and lethal mutagenesis. Favipiravir is effective on COVID-19 only at high doses. Current treatment scheme consists of 1600 mg bid on the first day followed by 600 mg bid po reaching a total dose of 8000 mg [14]. So far, no other cutaneous adverse effect of favipiravir has been reported despite its deposition in the respective tissues [15] and a call for awareness of potential favipiravir-induced phototoxicity [16].
Yellow fluorescence has also been observed in both lunula and nails after oral tetracycline therapy [17], and both phototoxicity [18] and photoonycholysis [19] have been an issue with oral tetracyclines.
UV-A exposure can have negative effects on eyes in both short-term and long-term use, and respective caution is warranted.
11.8 Microbiological Studies
Microbiologic studies are mandatory in inflammatory conditions of the scalp with scaling, crusting, and/or pustulation.
While in children fungal infections (tinea capitis) predominate, in the adult, bacterial infection with S. aureus is the most prominent. At times, repeated microbiologic studies are recommended, since with prolonged antibiotic treatments, typically in folliculitis decalvans, new and resistant pathogens may emerge, e.g., gram-negative folliculitis.
Diagnosis of fungal and bacterial skin infections requires swabs and test systems for direct visualization of pathogens (KOH preparation, gram stain), cultures and special tests for species identification, and the availability of the appropriate laboratory infrastructure.
The swab is the medical device used for the collection of biological samples from the body and allows for the transport and preservation of the sample. Some of the most common applications of swabs are for the isolation of microorganisms in culture media. A wad of absorbent material usually wound around one end of a small stick and used for removing material from an area. The purpose of microbiological sampling is to allow statements of density, types, and locations of microorganism which reside on the skin. Specimens for culture must be collected properly prior to the initiation particularly of systemic antimicrobial therapy to insure optimal conditions for the recovery of pathogens. The laboratory will identify isolates and perform antibiotic susceptibility testing where appropriate.
For diagnosis of tinea capitis infection and carrier state, different methods for obtaining samples have been reported, including the hairbrush, toothbrush, scalpel blade, gauze, carpet disc, or cotton swab methods. In one study, the hairbrush method was significantly found to be more effective in detecting dermatophyte fungi than the toothbrush and the cotton swab methods. There was also a statistically significant difference between the use of a single method and the combination of all other three methods [20].
A microbiological culture, or microbial culture, is a method of multiplying microbial organisms by letting them reproduce in predetermined culture medium under controlled laboratory conditions. Microbial cultures are used to determine the type of organism, its abundance in the sample being tested, or both. It is one of the primary diagnostic methods of microbiology and used as a tool to determine the cause of infectious disease.
Developing pure culture techniques is crucial to the observation of the specimen in question. The most common method to isolate individual cells and produce a pure culture is to prepare a streak plate. The streak plate method is a way to physically separate the microbial population and is done by spreading the inoculate back and forth with an inoculating loop over the solid agar plate. Upon incubation, colonies will arise and single cells will have been isolated from the biomass.
A pure or axenic culture is a population of cells or multicellular organisms growing in the absence of other species. For the purpose of gelling the microbial culture, the medium of agarose gel (agar) is used. Agar is a gelatinous substance derived from seaweed. Microbiological cultures can be grown in petri dishes of differing sizes that have a thin layer of agar-based growth medium. There are a variety of additives that can be added to agar before it is poured into a plate and allowed to solidify. Some types of bacteria can only grow in the presence of certain additives. Once a microorganism has been isolated in pure culture, it is necessary to preserve it in a viable state for further study and use.
Once the growth medium in the petri dish is inoculated with the desired bacteria, the plates are incubated at the optimal temperature for the growing of the selected bacteria, usually at 37 degrees Celsius for cultures from humans or animals, or lower for environmental cultures. After the desired level of growth is achieved, agar plates can be stored upside down in a refrigerator for an extended period of time to keep bacteria for future experimentations.
For single-celled eukaryotes, such as yeast, the isolation of pure cultures uses the same techniques as for bacterial cultures. Pure cultures of multicellular organisms are often more easily isolated by simply picking out a single individual to initiate a culture. This is a useful technique for pure culture of fungi.
Characteristics of culture rate of growth; colony morphology, including color of surface and reverse (underside); and microscopic morphologies of organisms allow for identification of specific fungal pathogens.
Microsporum canis is the most frequent reported zoophilic agent worldwide, while Trichophyton violaceum and Trichophyton tonsurans are the predominant anthropophilic agents. Over time, the frequency of these latter fungal infections has increased globally, and these fungi have become the major species globally. Anthropophilic transmission could be explained by the socioeconomic status of affected countries and population groups with associated risk factors and movement of populations importing new causes of infection to areas where they had not been encountered previously [21].
Microsporum canis is still the most common reported causative agent of tinea capitis in Europe. The countries reporting the highest incidence of M. canis infections are mainly in the Mediterranean but also bordering countries like Austria, Hungary, Germany, and Poland. Besides the increase in Microsporum-induced tinea capitis, there is a shift towards anthrophilic tinea capitis mainly in urban areas in Europe. The largest overall increase with anthropophilic dermatophytes has been noted with Trichophyton tonsurans mainly in the United Kingdom and with Trichophyton soudanense and Microsporum audouinii in France. The occurrence of anthropophilic infections seems to be geographically restricted and is possibly linked to the immigration from African countries. Children aged 3–7 years with no predilection of gender remain the most commonly affected, but recently an increase of tinea capitis has been observed in adults and in the elderly [22].
While Trichophyton rubrum has led to unprecedented worldwide suppression of other dermatophytes which had been predominant earlier as a causative agent of superficial dermatomycoses, in tinea capitis on the other hand, several other species of Trichophyton or Microsporum are dominant depending on the region or continent, and tinea capitis caused by T. rubrum is a rare event worldwide. The relative frequency of this causative agent in tinea capitis in children is under 1%. In adults, however, where tinea capitis occurs very infrequently indeed, the incidence of T. rubrum appears to exceed 10% [23].
On wet preparations of specimens on smeared and dried material, the fluorescent Calcofluor White stain, with or without potassium hydroxide (KOH), is far superior to the traditional KOH alone. It is particularly useful in detecting sparse mounts of fungi and exceptional for exhibiting certain morphologic structures of fungi that have been isolated on culture.
In culture, Microsporum canis is characterized by a moderate rate of growth, with maturity within 6–10 days, a whitish surface that is coarsely fluffy, hair to silky or fur-like, with yellow pigment at periphery and closely spaced radial grooves. Reverse is deep yellow and turns brownish yellow with age (Fig. 11.10a, b). Microscopic morphology is characterized by septate hyphae with numerous macroconidia, which are long, spindle-shaped, rough, and thick walls and characteristically taper to knob-like ends. Usually more than six compartments are seen in the macroconidia (Fig. 11.10c).
Microsporum audouinii exhibits a moderate rate of growth with maturity in 7–10 days. Colony morphology is characterized by a flat downy to silky, surface, with a radiating edge; it is garish or tannish white. Reverse is light salmon with reddish-brown center. Hyphae are septate with terminal chlamydoconidia that are often pointed at the end. Comb-like hyphae are commonly seen. This species is usually almost devoid of conidia but sometimes forms poorly shaped, abortive macroconidia or occasionally microconidia that are identical to those occurring in other species of the genus Microsporum.
Microsporum gallinae is a rare cause of tinea capitis and is more often seen as a cause of ringworm in chickens or other fowl. The rate of growth is moderate with maturity in 6–10 days. Colony morphology is characterized by slightly fluffy or satiny becoming pinkish with age. Reverse is yellow at first and later has a red pigment that diffuses into the medium (Fig. 11.11a, b). Hyphae are septate. Macroconidia have walls that are relatively thin and usually smooth. They contain four to ten cells, are blunt tipped, and are often distinctively curved with a tapering base. Microconidia are usually abundant.
Trichophyton mentagrophytes invades all parts of the body surface, including hair and nails, and is a common cause of tinea pedis. The rate of growth is moderate with maturity in 7–10 days. Colony morphology varies greatly. The surface may be buff and powdery or white and downy and may become pinkish or yellowish. The powdery form exhibits concentric radial folds. The colonies rapidly develop a dense fluff. The reverse is usually brownish tan but may be colorless, yellow, or red (Fig. 11.12a, b). The microscopic morphology is characterized by septate hyphae. Microconidia in powdery cultures are very round and clustered on branched conidiophores, in fluffy strains smaller, fewer in number, tear-shaped. Macroconidia are sometimes present, cigar-shaped, and thin-walled and have narrow attachments to hyphae and contain 1–6 cells. Coiled spiral hyphae are often seen.
Trichophyton rubrum is presently the most common dermatophyte infecting humans, primarily the skin and nails, and less frequently the beard or scalp. The rate of growth is moderately slow, with maturity within 14 days. Colony morphology is characterized by a granular or fluffy surface, white to buff. Reverse is deep red; occasionally it is brown, yellow-orange, or even colorless (Fig. 11.13a, b). Microscopic morphology is characterized by septate hyphae, tear-shaped microconidia that usually form singly all along the sides of the hyphae. Macroconidia may be abundant, rare, or absent; they are long, narrow, and thin-walled, with parallel sides (pencil-like), and have four to ten cells. They characteristically form directly on ends of thick hyphae. Microconidia characteristically form directly on macroconidia.
Trichophyton tonsurans is the principal etiologic pathogen of tinea capitis in the United States. Rate of growth in colony is moderately slow, with maturity in 12 days. Colony morphology is highly variable. Surface may be white greyish, yellow, rose, or brownish. Surface is usually suede-like, with many radial or concentric folds. Reverse is usually reddish-brown, sometimes yellow, or colorless (Fig. 11.14a, b). Microscopy morphology is characterized by septate hyphae, with many variably shaped microconidia all along the hyphae or on short conidiophores that are perpendicular to the parent hyphae. Microconidia are usually teardrop- or club-shaped but may be elongate or enlarge to round balloon forms. Macroconidia are rare and irregular in form.
Trichophyton soudanense is endemic in Central and West Africa and is increasingly being reported in Europe. Colony growth rate is slow with maturity within 15 days. Colonies show a yellow to orange, suede-like surface that is flat to folded, with a radiating fringe. Reverse is similar in color to the surface. Microscopy features are septate hyphae that often break up to form arthroconidia. Characteristically, branches form at both forward and backward angles to the parent hypha, often giving the appearance of barbed wire. No macroconidia are seen.
Trichophyton schoenleinii is the causative agent of favus. This notorious kind of tinea capitis used to be highly frequent globally, mainly in impoverished countries. Currently, it has become a historical form, except in some areas in Asia and Africa. The rate of growth in colony is slow with maturity within 15 days. Colony is whitish, waxy, or slightly downy, heaped, or folded (Fig. 11.15a). Growth is often submerged and splits the agar medium. Reverse is colorless or pale yellowish orange to tan. Microscopic morphology is characterized by septate, highly irregular, and knobby hyphae. The subsurface hyphae usually form characteristic antler-like branching structures with swollen tips resembling nail heads (Fig. 11.15b). Chlamydoconidia are numerous. Microconidia and macroconidia are absent.
Trichophyton verrucosum infects the scalp and beard and is usually contracted from cattle. Colony rate of growth is slow, with maturity in 14–21 days. The colonies are usually small, heaped, and button-like but sometimes flat. The texture is skin-like, waxy, or slightly downy, the color usually white, but can be gray or yellow. The reverse varies from nonpigmented to yellow. On Sabouraud dextrose agar at 37 °C forms hyphae with many chlamydoconidia often in chains and some antler-like branches.
Trichophyton violaceum is yet another fungal species that has increased in prevalence especially in urban populations of the United Kingdom and Europe with changes in immigration patterns and increases in international travel. It most commonly affects the scalp and hair. Colony growth rate is slow, with maturity in 14–21 days. Original cultures are waxy, wrinkled, heaped, and deep purplish red. Reverse is lavender to purple (Fig. 11.16). Microscopic morphology is characterized by tangled, branches, irregular, and granular hyphae, with intercalary chlamydoconidia. Microconidia and macroconidia are not usually seen on Sabouraud dextrose agar.
Candida albicans is the most common cause of candidiasis, which is an acute, subacute, or chronic infection involving any part of the body. Rate of growth in colony is rapid, with maturity in 3 days. Colonies are cream-colored, pasty, and smooth (Fig. 11.17). On routine primary media, yeast cells are round to oval.
Mucor spp. are known as common contaminants and are occasionally the etiologic agent of zygomycosis. Rate of growth in culture is rapid with maturity with 4 days. Colonies quickly covers agar surface with fluff resembling cotton candy (Fig. 11.18), white, and later turns gray or grayish brown. Reverse is white. Microscopic morphology is characterized by wide hyphae that are practically nonseptate. Sporangiophores are long and often branched and bear terminal round, spore-filled sporangia. The sporangial wall dissolves, scattering the round and slightly oblong spores.
Aspergillus spp. are opportunistic invaders, the most common molds to infect various sites in individuals with lowered resistance due to neutropenia and/or treatment with high-dose corticosteroids or cytotoxic drugs. Since the organisms are widespread in the environment, they are commonly found as contaminants in cultures. The rate of growth in culture is usually rapid, with maturity within 3 days. The colony surface is at first white and then any shade of green yellow, orange, brown, or black, depending on the species (Fig. 11.19). Texture is velvety or cottony. Reverse is usually white, goldish, or brown. Microscopic morphology is characterized by septate hyphae, an unbranched conidiophore arises from a specialized foot cell. The conidiophore is enlarged at the tip, forming a swollen vesicle. Vesicles are completely or partially covered with flask-shaped phialides, which may develop directly on the vesicle and produce chains of mostly round conidia.
Penicillium spp. are commonly considered as contaminants but found in a variety of diseases in which their etiologic significance is uncertain. Disseminated disease has been reported in severely immunocompromised patients. Many strains produce toxins. The rate of colony growth is rapid, with maturity within 4 days. The surface of colonies is at first white then becomes powdery and bluish green with a white border (Fig. 11.20). Reverse is usually white but may be red or brown. Microscopic morphology is characterized by septate hyphae with branched or unbranched conidiophores that have secondary branches. On those are flask-shaped phialides that bear unbranched chains of smooth or rough, round conidia. The entire structure forms the characteristic “penicillus” or “brush” appearance.
(a, b) Penicillium spp.: (a) the color of the surface is typically a dull dark green, (b) Penicillium chrysogenum (previously notatum). The fungal mycelium is seen, with new growth on the edges. It was the species which led Alexander Fleming (1881–1955) to discover quite by accident that this fungus inhibits the growth of bacteria, which subsequently led to the production of the first antibiotic penicillin
All clinical providers are recommended to ascertain the causative organism in fungal infection, either through fungal culture or newer methods, such as the polymerase chain reaction assay [24]. Those interested in involving in clinical mycology are encouraged to refer to the respective textbooks, such as Davise Honig Larone (author), Thomas J. Walsh (author), Randall T. Hayden (author), and Davise H. Larone (illustrator): Larone’s Medically Important Fungi: A Guide to Identification. ASM Books, 6th edition, 2018. This is an easy-to-use book that helps laboratory workers identify fungal pathogens under the microscope by their morphology and other readily identifiable features.
Polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies—complete or partial—of a specific DNA sample, allowing investigators to take a very small sample of DNA and amplify it or a part of it to a large enough amount to study in detail. PCR is now a common and often indispensable technique used in medical laboratory research for a broad variety of applications including biomedical research, criminal forensics, and detection of pathogens in nucleic acid tests for the diagnosis of infectious diseases.
Using PCR, copies of very small amounts of DNA sequences are exponentially amplified in a series of cycles of temperature changes. The majority of PCR methods rely on thermal cycling. Thermal cycling exposes reactants to repeated cycles of heating and cooling to permit different temperature-dependent reactions, specifically, DNA melting and enzyme-driven DNA replication. PCR employs two main reagents: primers which are short single-strand DNA fragments known as oligonucleotides that are a complementary sequence to the target DNA region and a DNA polymerase. In the first step of PCR, the two strands of the DNA double helix are physically separated at a high temperature in a process called nucleic acid denaturation. In the second step, the temperature is lowered, and the primers bind to the complementary sequences of DNA. The two DNA strands then become templates for DNA polymerase to enzymatically assemble a new DNA strand from free nucleotides, the building blocks of DNA. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the original DNA template is exponentially amplified. Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the thermophilic bacterium Thermus aquaticus. If the polymerase used was heat susceptible, it would denature under the high temperatures of the denaturation step. Before the use of Taq polymerase, DNA polymerase had to be manually added every cycle, which was a tedious and costly process.
11.9 Blood Tests
Diagnostic tests are useful when the probability of a disease being present is neither high nor low, since high degree of clinical certainty overrides the uncertainty of the laboratory data. The greater the number of different tests done, the greater the risk of getting false-positive or irrelevant leads. The possibilities for laboratory errors increase in the automated multiple-screen procedures. Therefore, laboratory testing must be kept sharply focused. Clinical suspicion is the determinant, and knowledge of clinical dermatology is the prerequisite for combining medical sense with economic sense in requesting laboratory tests.
Elevations in the white blood count (WBC) are often associated with infection, though many viral infections are associated with leukopenia. It is important to assess the WBC differential, given that different classes of microbes are associated with various leukocyte types. For example, bacteria are associated with an increase in polymorphonuclear neutrophils, often with elevated levels of earlier developmental forms such as bands; viruses are associated with an increase in lymphocytes, and certain parasites are associated with an increase in eosinophils.
The erythrocyte sedimentation rate (ESR) and the C-reactive protein (CRP) level are indirect and direct measures of the acute-phase response, respectively, that can be used to assess a patient’s general level of inflammation. Moreover, these markers can be followed serially over time to monitor disease progress/resolution. It is noteworthy that the ESR changes relatively slowly, and its measurement more often than weekly usually is not useful: in contrast, CRP concentrations change rapidly, and daily measurements can be useful in the appropriate context. Although these markers are sensitive indicators of inflammation, neither is very specific. An extremely elevated ESR (>100 mm/h) has a 90% predictive value for a serious underlying disease.
The technology of infectious disease serology assays has been thus evolving ever since the Widal test more than 100 years ago to detect typhoid fever in serum from patients with fever suspected for Salmonella typhi infection [25], and it has been advancing rapidly in the last decades. The application of infectious disease serological assays is extremely broad since theoretically it can be developed for every pathogen. Despite the evolving technology in serological assays, serology is seldom used as the sole diagnostic tool. Only for a few bacterial infections, such as syphilis and disseminated manifestations of Lyme disease, are serological assays still used as the primary test for establishing the diagnosis.
In clinical virology, serology is mostly used to determine the stage of infection (acute versus past) by detecting the presence of the IgM antibodies, or by showing significant changes in antibody titers (follow-up serum samples are needed), or by avidity testing (IgG antibody produced early in the infection showing low binding strength between antibodies and virus).
Syphilis may be difficult to diagnose clinically, particularly during early infection. Confirmation is either via direct visual inspection using dark field microscopy or blood tests. Blood tests are more commonly used, as they are easier to perform.
A non-treponemal test is a blood test for diagnosis of infection with syphilis. Non-treponemal tests are an indirect method in that they detect biomarkers that are released during cellular damage that occurs from the syphilis spirochete. Syphilitic infection leads to the production of nonspecific antibodies that react to cardiolipin. This reaction is the foundation of the non-treponemal assays such as the VDRL (Venereal Disease Research Laboratory) test and rapid plasma reagin (RPR) test that are flocculation-type tests that use an antigen-antibody interaction. The complexes remain suspended in solution and therefore visible due to the lipid-based antigens. These have replaced the original non-treponemal test, the Wassermann test. The non-treponemal tests measure immunoglobulins G (IgG) and M (IgM) anti-lipid antibodies formed by the host in response both to lipoidal material released from damaged host cells early in infection and to lipid from the cell surfaces of the treponeme itself. These non-treponemal tests are widely used for qualitative syphilis screening, since these tests are relatively simple to perform and interpret and can allow rapid return of results and are very cheap. However, their usefulness is limited by decreased sensitivity in early primary syphilis and during late syphilis. In addition, with non-treponemal tests, false-positive reactions can occur for a large number of reasons, the most common of which is other infections or connective tissue diseases, such as borreliosis and lupus erythematosus, respectively. Moreover, these tests may show false-negative when the patient’s antibody titer is very high due to a so-called prozone effect. Because of the issues with false positives, confirmation with a second treponemal test that is specific for T. pallidum antibodies is recommended [26].
In contrast, the treponemal tests look for antibodies that are a direct result of the infection thus, anti-treponeme IgG and IgM.
The fluorescent treponemal antibody absorption (FTA-ABS) test is a diagnostic test for syphilis. Using antibodies specific for the Treponema pallidum species, such tests would be assumed to be more specific than non-treponemal testing such as VDRL but have been shown repeatedly to be sensitive but not specific for the diagnosis of neurosyphilis in CSF. FTA is nearly 100% sensitive in CSF. Negative FTA in CSF and exclude neurosyphilis. In addition, FTA-ABS turns positive earlier and remains positive longer than VDRL. This test is not useful for following therapy, because it does not wane with successful treatment of the disease and will continue to be positive for many years after primary exposure.
The Treponema pallidum particle agglutination assay (TPPA test) is an indirect agglutination assay used for detection and titration of antibodies against Treponema pallidum. In the test, gelatin particles are sensitized with T. pallidum antigen. Patient serum is mixed with the reagent containing the sensitized gelatin particles. The particles aggregate to form clumps when the patient serum is positive for syphilis. In other words, the patient’s serum contains antibodies to T. pallidum. A negative test shows no clumping of gelatin particles. For primary syphilis, TPPA has a sensitivity of 85–100% and a specificity of 98–100%. In secondary and late-latent syphilis, TPPA has a sensitivity of 98–100%.
A similar specific treponemal test for syphilis is the Treponema pallidum hemagglutination assay (TPHA). TPHA is an indirect hemagglutination assay used for the detection and titration of antibodies against Treponema pallidum. In the test, erythrocytes are sensitized with antigens from T. pallidum. The cells then aggregate on the surface of a test dish if exposed to the serum of a patient with syphilis. It is used as a confirmatory test for syphilis infection. A negative test result shows a tight button or spot of red blood cells on the surface of the test dish. Often a plastic test plate containing many small wells is used as the test dish so that many patients may be tested at the same time, but their results can be kept separate from each other.
The traditional testing algorithm for syphilis begins testing with the non-treponemal test. If the non-treponemal test is reactive, a treponemal test is then used to confirm syphilis infection.
The reverse testing algorithm for syphilis begins testing with a treponemal test. If this test is reactive, a non-treponemal test is performed. When the non-treponemal test is nonreactive, a second treponemal test is performed to determine if the first treponemal test was a false positive. The second treponemal test performed must be different than the initial treponemal test. The reverse testing algorithm has been in place since 2009. This algorithm is attractive to laboratories that have a high testing volume because it reduces the amount of manual labor conducted for the non-treponemal tests.
Because the antibodies detected in treponemal tests usually remain detectable for life, even after successful treatment, the non-treponemal titer (RPR or VDRL) must be used to monitor for a reinfection with syphilis. An increase in titer of two dilutions represents reinfection with Treponema pallidum.
Two-tiered serological testing is performed for differential diagnosis of Borrelia infection (Lyme disease). The first-tier tests detect specific antibodies (IgM and IgG together or separately) and include enzyme-linked immunoassays (EIA) and immunofluorescent assays. Positive results for first-tier tests are confirmed using second-tier testing. The second tier consists of standardized immunoblotting, by using either Western blots or blots striped with diagnostically important purified antigens. Positive results for second-tier tests are confirmatory for the presence of Borrelia infection.
The recommended two-tiered serology for Lyme disease begins with an EIA. If the EIA is positive or equivocal, immunoblotting should be performed. Both a positive or equivocal EIA and a sufficient number of immunoblot bands (≥2 of 3 IgM or ≥5 of 10 IgG) are required for a positive test. Clinicians interpreting test results should not focus on individual positive bands as these have no diagnostic value on their own. Patients with Lyme disease symptoms (>30 days) require positive IgG immunoblot results to confirm diagnosis [27].
QuantiFERON-TB Gold (QFT) is a simple blood test that aids in the detection of Mycobacterium tuberculosis. QFT is an interferon-gamma (IFN-γ) release assay, commonly known as an IGRA, and is a modern alternative to the tuberculin skin test (TST, PPD, or Mantoux). Unlike the TST, QFT is a controlled laboratory test that requires only one patient visit and is unaffected by previous Bacille Calmette-Guerin (BCG) vaccination. QFT is highly specific and sensitive: a positive result is strongly predictive of true infection with M. tuberculosis. However, like the TST and other IGRAs, QFT cannot distinguish between active tuberculosis disease and latent tuberculosis infection and is intended for use with risk assessment, radiography, and other medical and diagnostic evaluations. Like any diagnostic aid, QFT cannot replace clinical judgment.
Considering the emerging infectious diseases, Fischer et al. reviewed the use of serological tests to diagnose such as Zika, Dengue fever, and Chikungunya [28].
COVID-19 testing involves analyzing samples to assess the current or past presence of SARS-CoV-2. The two main types of tests detect either the presence of the virus or antibodies produced in response to infection.
Molecular tests for viral presence through its molecular components are used to diagnose individual cases and to allow public health authorities to trace and contain outbreaks. There are multiple types of tests that look for the virus by detecting the presence of the virus’s RNA. As of 2021, the most common form of molecular test is the reverse transcription polymerase chain reaction (RT-PCR) test [29]. Antigen tests look for antigen proteins from the viral surface. In the case of a coronavirus, these are usually proteins from the surface spikes. SARS-CoV-2 antigens can be detected before onset of COVID-19 symptoms (as soon as SARS-CoV-2 virus particles) with more rapid test results (Fig. 11.21a, b) but with less sensitivity than PCR tests for the virus [30].
Antibody tests (serology immunoassays) instead show whether someone once had the disease [31]. They are less useful for diagnosing current infections because antibodies may not develop for weeks after infection. It is used to assess disease prevalence, which aids the estimation of the infection fatality rate [32]. Test analysis is often performed in automated, high-throughput, medical laboratories. Rapid self-tests and point-of-care testing are also available and can offer a faster and less expensive method to test for the virus although with a lower accuracy.
11.10 Imaging
Imaging provides an important adjunct to the physical examination, allowing evaluation for lymphadenopathy in regions that are not externally accessible, such as the mediastinum, or intra-abdominal sites, and assessment of internal organs for evidence of infection, particularly the lungs, the liver, the kidneys, and the brain.
Ultimately, the dermatologist participates with the other medical disciplines in the diagnosis and treatment of infections as they may relate to systemic disease. Specifically, infection multidisciplinary team meetings allow for detailed and combined review of the history, examination, and imaging findings of a patient, which leads to improved use of radiological investigations and patient management [33]. Particularly in the wake of the COVID-19 pandemic, the importance of interdisciplinary communication has become evident both in research and infectious disease management.
References
Heneghan C, Glasziou P, Thompson M, Rose P, Balla J, Lasserson D, Scott C, Perera R. Diagnostic strategies used in primary care. BMJ. 2009;338:b946.
Pinheiro AMC, Lobato LA, Varella TCN. Dermoscopy findings in tinea capitis: case report and literature review. An Bras Dermatol. 2012;87(2):313–4.
Gómez-Moyano E, Martínez Pilar L, Vera Casaño A, Martín González T, Moyano Almagro B, Fernandez Ballesteros MD, Martínez Garcia S, Godoy Diaz DJ. Correlation between dermoscopy and direct microscopy in tinea barbae. J Mycol Med. 2021;31(1):101104.
Piraccini BM, Broccoli A, Starace M, Gaspari V, D'Antuono A, Dika E, Patrizi A. Hair and scalp manifestations in secondary syphilis: epidemiology, clinical features and trichoscopy. Dermatology. 2015;231(2):171–6.
Bakos RM, Bakos L. Dermoscopy for diagnosis of pediculosis capitis. J Am Acad Dermatol. 2007;57(4):727–8.
Haliasos EC, Kerner M, Jaimes-Lopez N, Rudnicka L, Zalaudek I, Malvehy J, Hofmann-Wellenhof R, Braun RP, Marghoob AA. Dermoscopy for the pediatric dermatologist part I: dermoscopy of pediatric infectious and inflammatory skin lesions and hair disorders. Pediatr Dermatol. 2013;30(2):163–71.
Durdu M, Errichetti E, Eskiocak AH, Ilkit M. High accuracy of recognition of common forms of folliculitis by dermoscopy: an observational study. J Am Acad Dermatol. 2019;81(2):463–71.
Uchiyama M, Harada K, Tobita R, Irisawa R, Tsuboi R. Histopathologic and dermoscopic features of 42 cases of folliculitis decalvans: a case series. J Am Acad Dermatol. 2021;85(5):1185–93.
Saceda-Corralo D, Moreno-Arrones OM, Rodrigues-Barata R, Rubio-Lombraña M, Mir-Bonafé JF, Morales-Raya C, Miguel-Gómez L, Hermosa-Gelbard Á, Jaén-Olasolo P, Vañó-Galván S. Trichoscopy activity scale for folliculitis decalvans. J Eur Acad Dermatol Venereol. 2020;34(2):e55–7.
Trüeb RM, Dias MFRG. A comment on trichoscopy. Int J Trichology. 2018;10(4):147–9.
Rezende HD, Orquiza B, dos Santos B, Elias M, Dinato SLM, Dias MFRG, Trüeb RM. How to prepare a trichogram with transparent enamel base coat? Surg Cosmet Dermatol. 2021;13:20210037.
Kayıran MA, Cebeci F, Erdemir VA, Aksoy H, Akdeniz N, Gürel MS. Fluorescence of nails and hair on Wood's lamp examination in Covid pandemic; undefined effect of Favipiravir in humans. Dermatol Ther. 2021;34(1):e14740.
Yaghoubi A, Amel Jamehdar S, Movaqar A, Milani N, Soleimanpour S. An effective drug against COVID-19: reality or dream? Expert Rev Respir Med. 2020;15:505–18.
Kaptein SJF, Jacobs S, Langendries L, et al. Favipiravir at high doses has potent antiviral activity in SARS-CoV-2-infected hamsters, whereas hydroxychloroquine lacks activity. Proc Natl Acad Sci U S A. 2020;117(43):26955–65.
Martinez-Lopez A, Cuenca-Barrales C, Montero-Vilchez T, Molina-Leyva A, Arias-Santiago S. Review of adverse cutaneous reactions of pharmacologic interventions for COVID-19: a guide for the dermatologist. J Am Acad Dermatol. 2020;83(6):1738–48.
Turan Ç, Metin N, Utlu Z, Yıldız TT, Sakat SC. Evaluation of the frequency and intensity of favipiravir-associated yellow-green fluorescence in lunulae, hair, and face. J Cosmet Dermatol. 2022;21(3):1199–207.
Hendricks AA. Yellow lunulae with fluorescence after tetracycline therapy. Arch Dermatol. 1980;116(4):438–40.
Smith EL, al Raddadi A, al Ghamdi F, Kutbi S. Tetracycline phototoxicity. Br J Dermatol. 1995;132(2):316–7.
Lasser AE, Steiner MM. Tetracycline photo-onycholysis. Pediatrics. 1978;61(1):98–9.
Akbaba M, Ilkit M, Sutoluk Z, Ates A, Zorba H. Comparison of hairbrush, toothbrush and cotton swab methods for diagnosing asymptomatic dermatophyte scalp carriage. J Eur Acad Dermatol Venereol. 2008r;22(3):356–62.
Rodríguez-Cerdeira C, Martínez-Herrera E, Szepietowski JC, Pinto-Almazán R, Frías-De-León MG, Espinosa-Hernández VM, Chávez-Gutiérrez E, García-Salazar E, Vega-Sánchez DC, Arenas R, Hay R, Saunte DM. A systematic review of worldwide data on tinea capitis: analysis of the last 20 years. J Eur Acad Dermatol Venereol. 2021;35(4):844–83.
Ginter-Hanselmayer G, Weger W, Ilkit M, Smolle J. Epidemiology of tinea capitis in Europe: current state and changing patterns. Mycoses. 2007;50(Suppl 2):6–13.
Schwinn A, Ebert J, Bröcker EB. Frequency of Trichophyton rubrum in tinea capitis. Mycoses. 1995;38(1–2):1–7.
Le TK, Cohen BA. Tinea capitis: advances and a needed paradigm shift. Curr Opin Pediatr. 2021;33(4):387–91.
Olopoenia LA, King AL. Widal agglutination test—100 years later: still plagued by controversy. Postgrad Med J. 2000;76(Internet):80–4.
Ratnam S. The laboratory diagnosis of syphilis. Can J Infect Dis Med Microbiol. 2005;16(1):45–51.
Lantos PM, Auwaerter PG, Nelson CA. Lyme disease serology. JAMA. 2016;315(16):1780–1.
Fischer C, Jo WK, Haage V, Moreira-Soto A, Ferreira de Oliveira-Filho E, Drexler JF. Challenges towards serologic diagnostics of emerging arboviruses. Clin Microbiol Infect. 2021;27:1221–9.
Habibzadeh P, Mofatteh M, Silawi M, Ghavami S, Faghihi MA. Molecular diagnostic assays for COVID-19: an overview. Crit Rev Clin Lab Sci. 2021;58(6):385–98.
Guglielmi G. Fast coronavirus tests: what they can and can't do. Nature. 2020;585(7826):496–8.
Kubina R, Dziedzic A. Molecular and serological tests for COVID-19 a comparative review of SARS-CoV-2 coronavirus laboratory and point-of-care diagnostics. Diagnostics. 2020;10(6):434.
Abbasi J. The promise and peril of antibody testing for COVID-19. JAMA. 2020;323(19):1881–3.
Fletcher TE, Ali H, Ryall C, Beeching NJ, Joekes E, Lewthwaite P. The benefits of an infectious disease/radiology multidisciplinary team meeting. J Infect. 2012;65(4):363–5.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Nature Switzerland AG
About this chapter
Cite this chapter
Trüeb, R.M., Dutra Rezende, H., Gavazzoni Dias, M.F.R. (2023). Diagnostic Techniques. In: Trüeb, R.M., Dutra Rezende, H., Gavazzoni Dias, M.F.R. (eds) Hair in Infectious Disease. Springer, Cham. https://doi.org/10.1007/978-3-031-30754-6_11
Download citation
DOI: https://doi.org/10.1007/978-3-031-30754-6_11
Published:
Publisher Name: Springer, Cham
Print ISBN: 978-3-031-30753-9
Online ISBN: 978-3-031-30754-6
eBook Packages: MedicineMedicine (R0)