Genetic epidemiology of Sarcoptes scabiei (Acari: Sarcoptidae) in northern Australia

Utilising three hypervariable microsatellite markers we have previously shown that scabies mites on people are genetically distinct from those on dogs in sympatric populations in northern Australia. This had important ramifications on the formulation of public health control policies. In contrast phylogenetic analyses using mitochondrial markers on scabies mites infecting multiple animal hosts elsewhere in the world could not differentiate any genetic variation between mite haplotype and host species. Here we further analyse the intra-specific relationship of Sarcoptes scabiei var. hominis with S. scabiei var. canis by using both mitochondrial DNA and an expanded nuclear microsatellite marker system. Phylogenetic studies using sequences from the mitochondrial genes coding for 16S rRNA and Cytochrome Oxidase subunit I demonstrated significant relationships between S. scabiei MtDNA haplotypes, host species and geographical location. Multi-locus genotyping using 15 microsatellite markers substantiated previous data that gene flow between scabies mite populations on human and dog hosts is extremely rare in northern Australia. These data clearly support our previous contention that control programs for human scabies in endemic areas with sympatric S. scabiei var. hominis and var. canis populations must focus on human-to-human transmission. The genetic division of dog and human derived scabies mites also has important implications in vaccine and diagnostic test development as well as the emergence and monitoring of drug resistance in S. scabiei in northern Australia.     

The Effect of Insecticide Synergists on the Response of Scabies Mites to Pyrethroid Acaricides

Background

Permethrin is the active component of topical creams widely used to treat human scabies. Recent evidence has demonstrated that scabies mites are becoming increasingly tolerant to topical permethrin and oral ivermectin. An effective approach to manage pesticide resistance is the addition of synergists to counteract metabolic resistance. Synergists are also useful for laboratory investigation of resistance mechanisms through their ability to inhibit specific metabolic pathways.

Methodology/Principal Findings

To determine the role of metabolic degradation as a mechanism for acaricide resistance in scabies mites, PBO (piperonyl butoxide), DEF (S,S,S-tributyl phosphorotrithioate) and DEM (diethyl maleate) were first tested for synergistic activity with permethrin in a bioassay of mite killing. Then, to investigate the relative role of specific metabolic pathways inhibited by these synergists, enzyme assays were developed to measure esterase, glutathione S-transferase (GST) and cytochrome P450 monooxygenase (cytochrome P450) activity in mite extracts. A statistically significant difference in median survival time of permethrin-resistant Sarcoptes scabiei variety canis was noted when any of the three synergists were used in combination with permethrin compared to median survival time of mites exposed to permethrin alone (p<0.0001). Incubation of mite homogenates with DEF showed inhibition of esterase activity (37%); inhibition of GST activity (73%) with DEM and inhibition of cytochrome P450 monooxygenase activity (81%) with PBO. A 7-fold increase in esterase activity, a 4-fold increase in GST activity and a 2-fold increase in cytochrome P450 monooxygenase activity were observed in resistant mites compared to sensitive mites.

Conclusions

These findings indicate the potential utility of synergists in reversing resistance to pyrethroid-based acaricides and suggest a significant role of metabolic mechanisms in mediating pyrethroid resistance in scabies mites.     

Three mutations identified in the voltage-sensitive sodium channel alpha-subunit gene of permethrin-resistant human head lice reduce the permethrin sensitivity of house fly Vssc1 sodium channels expressed in Xenopus oocytes

Point mutations in the para-orthologous sodium channel alpha-subunit of the head louse (M815I, T917I, and L920F) are associated with permethrin resistance and DDT resistance. These mutations were inserted in all combinations using site-directed mutagenesis at the corresponding amino acid sequence positions (M827I, T929I, and L932F) of the house fly para-orthologous voltage-sensitive sodium channel alpha-subunit (Vssc1(WT)) gene and heterologously co-expressed with the sodium channel auxiliary subunit of house fly (Vsscbeta) in Xenopus oocytes. The double mutant possessing M827I and T929I (Vssc1(MITI)/Vsscbeta) caused a approximately 4.0mV hyperpolarizing shift and the triple mutant, Vssc1(MITILF)/Vsscbeta, caused a approximately 3.2mV depolarizing shift in the voltage dependence of activation curves. Vssc1(MITI)/Vsscbeta, Vssc1(TILF)/Vsscbeta, and Vssc1(MITILF)/Vsscbeta caused depolarizing shifts ( approximately 6.6, approximately 7.6, and approximately 8.8mV, respectively) in the voltage dependence of steady-state inactivation curves. The M827I and L932F mutations reduced permethrin sensitivity when expressed alone but the T929I mutation, either alone or in combination, virtually abolished permethrin sensitivity. Thus, the T929I mutation is the principal cause of permethrin resistance in head lice. Comparison of the expression rates of channels containing single, double and triple mutations with that of Vssc1(WT)/Vsscbeta channels indicates that the M827I mutation may play a role in rescuing the decreased expression of channels containing T929I.     

Scabies: molecular perspectives and therapeutic implications in the face of emerging drug resistance.

Limited effective treatments, coupled with recent observations of emerging drug resistance to oral ivermectin and 5% permethrin, raise concerns regarding the future control of scabies, especially in severe cases and in endemic areas where repeated community treatment programs are in place. There is consequently an urgent need to define molecular mechanisms of drug resistance in scabies mites and to develop and assess alternative therapeutic options, such as tea tree oil, in the event of increasing treatment failure. Molecular studies on scabies mites have, until recently, been restricted; however, recent advances are providing new insights into scabies mite biology and genetic mechanisms underlying drug resistance. These may assist in overcoming many of the current difficulties in monitoring treatment efficacy and allow the development of more sensitive tools for monitoring emerging resistance.     

Acaricidal Activity of Eugenol Based Compounds against Scabies Mites

Backgound

Human scabies is a debilitating skin disease caused by the “itch mite” Sarcoptes scabiei. Ordinary scabies is commonly treated with topical creams such as permethrin, while crusted scabies is treated with topical creams in combination with oral ivermectin. Recent reports of acaricide tolerance in scabies endemic communities in Northern Australia have prompted efforts to better understand resistance mechanisms and to identify potential new acaricides. In this study, we screened three essential oils and four pure compounds based on eugenol for acaricidal properties.

Methodology/Principal Findings

Contact bioassays were performed using live permethrin-sensitive S. scabiei var suis mites harvested from pigs and permethrin-resistant S. scabiei var canis mites harvested from rabbits. Results of bioassays showed that clove oil was highly toxic against scabies mites. Nutmeg oil had moderate toxicity and ylang ylang oil was the least toxic. Eugenol, a major component of clove oil and its analogues –acetyleugenol and isoeugenol, demonstrated levels of toxicity comparable to benzyl benzoate, the positive control acaricide, killing mites within an hour of contact.

Conclusions

The acaricidal properties demonstrated by eugenol and its analogues show promise as leads for future development of alternative topical acaricides to treat scabies.     

Treatment of scabies infestations

Scabies is an intensely pruritic disorder induced by an immune allergic response to infestation of the skin by the mite Sarcoptes scabiei. The biology of the mite, the clinical aspects and diagnosis of scabies infestations as well as the treatment of choice with 5% permethrin dermal cream and the use of scabicides based on other chemical substances are reviewed.     

Evaluation of the poultry red mite, Dermanyssus gallinae (Acari: Dermanyssidae) susceptibility to some acaricides in field populations from Italy

Red mite field populations from seven naturally infested Italian caged laying poultry farms were investigated for their susceptibility to acaricide formulations available on the market, containing amitraz, carbaryl and permethrin. A minimum of 3,000 mites of all stages were collected from each farm and were tested with five acaricide concentrations (5, 10, 20, 50, 100%) plus an untreated control (0%). Field red mite populations were found to be tolerant even with the highest concentrations with carbaryl and permethrin for six (86%) and three (42%) of the investigated farms, respectively (P < 0.05). Furthermore, six (86%) of the investigated farms showed a red mite population susceptible to amitraz at any concentration. Out of the seven field populations tested with amitraz, one population is becoming less tolerant whereas another was the most tolerant to carbaryl and permethrin at any concentration. Data show that the lack of effectiveness of some acaricides is spreading in Europe and call for the adoption of alternative management strategies to avoid development of resistance.     

Analysis of Sarcoptes scabiei finds no evidence of infection with Wolbachia

The endosymbiont Wolbachia has been detected in a range of filarial nematodes and parasitic mites and is known to affect host reproductive compatibility and potentially evolutionary processes. PCR of Wolbachia surface protein (wsp), ftsZ and 16SrRNA genes from individual Sarcoptes scabiei mites obtained from a series of individual hosts, and database searches of an S. scabiei var. hominis EST library failed to detect Wolbachia genes. Therefore, Wolbachia appears not to be involved in the genetic subdivision observed between varieties of host-associated S. scabiei or, involved in the inflammatory disease pathogenesis of scabies unlike its activity in filarial infection.     

Functional analysis and localisation of a delta-class glutathione S-transferase from Sarcoptes scabiei

The mite Sarcoptes scabiei causes sarcoptic mange, or scabies, a disease that affects both animals and humans worldwide. Our interest in S. scabiei led us to further characterise a glutathione S-transferase. This multifunctional enzyme is a target for vaccine and drug development in several parasitic diseases. The S. scabiei glutathione S-transferase open reading frame reported here is 684 nucleotides long and yields a protein with a predicted molecular mass of 26 kDa. Through phylogenetic analysis the enzyme was classified as a delta-class glutathione S-transferase, and our paper is the first to report that delta-class glutathione S-transferases occur in organisms other than insects. The recombinant S. scabiei glutathione S-transferase was expressed in Escherichia coli via three different constructs and purified for biochemical analysis. The S. scabiei glutathione S-transferase was active towards the substrate 1-chloro-2,4-dinitrobenzene, though the positioning of fusion partners influenced the kinetic activity of the enzyme. Polyclonal antibodies raised against S. scabiei glutathione S-transferase specifically localised the enzyme to the integument of the epidermis and cavities surrounding internal organs in adult parasites. However, some minor staining of parasite intestines was observed. No staining was seen in host tissues, nor could we detect any antibody response against S. scabiei glutathione S-transferase in sera from naturally S. scabiei infected dogs or pigs. Additionally, the polyclonal sera raised against recombinant S. scabiei glutathione S-transferase readily detected a protein from mites, corresponding to the predicted size of native glutathione S-transferase.     

Scabies Mites Alter the Skin Microbiome and Promote Growth of Opportunistic Pathogens in a Porcine Model

Background

The resident skin microbiota plays an important role in restricting pathogenic bacteria, thereby protecting the host. Scabies mites (Sarcoptes scabiei) are thought to promote bacterial infections by breaching the skin barrier and excreting molecules that inhibit host innate immune responses. Epidemiological studies in humans confirm increased incidence of impetigo, generally caused by Staphylococcus aureus and Streptococcus pyogenes, secondary to the epidermal infestation with the parasitic mite. It is therefore possible that mite infestation could alter the healthy skin microbiota making way for the opportunistic pathogens. A longitudinal study to test this hypothesis in humans is near impossible due to ethical reasons. In a porcine model we generated scabies infestations closely resembling the disease manifestation in humans and investigated the scabies associated changes in the skin microbiota over the course of a mite infestation.

Methodology/Principal Findings

In a 21 week trial, skin scrapings were collected from pigs infected with S. scabies var. suis and scabies-free control animals. A total of 96 skin scrapings were collected before, during infection and after acaricide treatment, and analyzed by bacterial 16S rDNA tag-encoded FLX-titanium amplicon pyrosequencing. We found significant changes in the epidermal microbiota, in particular a dramatic increase in Staphylococcus correlating with the onset of mite infestation in animals challenged with scabies mites. This increase persisted beyond treatment from mite infection and healing of skin. Furthermore, the staphylococci population shifted from the commensal S. hominis on the healthy skin prior to scabies mite challenge to S. chromogenes, which is increasingly recognized as being pathogenic, coinciding with scabies infection in pigs. In contrast, all animals in the scabies-free cohort remained relatively free of Staphylococcus throughout the trial.

Conclusions/Significance

This is the first experimental in vivo evidence supporting previous assumptions that establishment of pathogens follow scabies infection. Our findings provide an explanation for a biologically important aspect of the disease pathogenesis. The methods developed from this pig trial will serve as a guide to analyze human clinical samples. Studies building on this will offer implications for development of novel intervention strategies against the mites and the secondary infections.     

A Single Crossing-Over Event in Voltage-Sensitive Na+ Channel Genes May Cause Critical Failure of Dengue Mosquito Control by Insecticides

The voltage-sensitive sodium (Na⁺) channel (Vssc) is the target site of pyrethroid insecticides. Pest insects develop resistance to this class of insecticide by acquisition of one or multiple amino acid substitution(s) in this channel. In Southeast Asia, two major Vssc types confer pyrethroid resistance in the dengue mosquito vector Aedes aegypti, namely, S989P+V1016G and F1534C. We expressed several types of Vssc in Xenopus oocytes and examined the effect of amino acid substitutions in Vssc on pyrethroid susceptibilities. S989P+V1016G and F1534C haplotypes reduced the channel sensitivity to permethrin by 100- and 25-fold, respectively, while S989P+V1016G+F1534C triple mutations reduced the channel sensitivity to permethrin by 1100-fold. S989P+V1016G and F1534C haplotypes reduced the channel sensitivity to deltamethrin by 10- and 1-fold (no reduction), respectively, but S989P+V1016G+F1534C triple mutations reduced the channel sensitivity to deltamethrin by 90-fold. These results imply that pyrethroid insecticides are highly likely to lose their effectiveness against A. aegypti if such a Vssc haplotype emerges as the result of a single crossing-over event; thus, this may cause failure to control this key mosquito vector. Here, we strongly emphasize the importance of monitoring the occurrence of triple mutations in Vssc in the field population of A. aegypti.     

An Aspartic Protease of the Scabies Mite Sarcoptes scabiei Is Involved in the Digestion of Host Skin and Blood Macromolecules

Background

Scabies is a disease of worldwide significance, causing considerable morbidity in both humans and other animals. The scabies mite Sarcoptes scabiei burrows into the skin of its host, obtaining nutrition from host skin and blood. Aspartic proteases mediate a range of diverse and essential physiological functions such as tissue invasion and migration, digestion, moulting and reproduction in a number of parasitic organisms. We investigated whether aspartic proteases may play role in scabies mite digestive processes.

Methodology/Principle Findings

We demonstrated the presence of aspartic protease activity in whole scabies mite extract. We then identified a scabies mite aspartic protease gene sequence and produced recombinant active enzyme. The recombinant scabies mite aspartic protease was capable of digesting human haemoglobin, serum albumin, fibrinogen and fibronectin, but not collagen III or laminin. This is consistent with the location of the scabies mites in the upper epidermis of human skin.

Conclusions/Significance

The development of novel therapeutics for scabies is of increasing importance given the evidence of emerging resistance to current treatments. We have shown that a scabies mite aspartic protease plays a role in the digestion of host skin and serum molecules, raising the possibility that interference with the function of the enzyme may impact on mite survival.     

Persistence and effectiveness of pyrethroids in plastic strips against Varroa jacobsoni (Acari: Varroidae) and mite resistance in a Mediterranean area

An apiary trial was conducted in 1997 in Sardinia, Italy, to verify the effectiveness of fluvalinate in polyvinyl chloride strips and flumethrin in polyethylene strips against Varroa jacobsoni Oudemans. Two indices to evaluate the efficacy of the treatments were adopted: percentage change in mite infestation of worker-sealed brood cells considering only treated hives and percentage change in mite mortality, and the natural variation in mite populations recorded in control hives during the trial. All acaricide treatments reduced the level of mite infestation of both sealed brood and adult bees. However, their effectiveness was slightly reduced in comparison to previous studies because of mite resistance phenomena. Portions of polyethylene strips of flumethrin from treated hives were sampled weekly to determine acaricide persistence using gas chromatography. After 4 wk, a slight reduction (approximately 9%) of the active ingredient content was observed. A laboratory bioassay also was performed to establish the resistance of adult female mites to fluvalinate. Mites were sampled from the experimental apiary and from various Sardinian apiaries which had primarily been subjected to fluvalinate applications in plastic strips or wood inserts for years. Mite resistance varied from 0 to 96%, depending on the acaricide management adopted. The lowest resistance level occurred in an apiary where pyrethroids had never been used, whereas the highest level occurred in an apiary, with intensive use of fluvalinate in wood inserts.     

Molecular characterisation of a pH-gated chloride channel from <Emphasis Type="Italic">Sarcoptes scabiei</Emphasis>

Reports of ivermectin resistance in scabies mites raise concerns regarding the sustainability of mass intervention programs for scabies worldwide and for the treatment of crusted scabies. Ligand gated ion channels (LGICs) are the primary targets of ivermectin in invertebrates. We report the molecular characterisation of SsCl—a novel LGIC from Sarcoptes scabiei var. hominis. While SsCl shows sequence similarity to other LGICs, phylogenetic analysis does not suggest strong homology to conventional glutamate, histamine or GABA gated channels. Instead, it is most similar to Drosophila pH-sensitive and group 1 clades. When expressed in Xenopus oocytes, SsCl forms a homomeric, pH-gated chloride channel that is irreversibly activated by ivermectin. These results provide the first confirmation that this group of LGIC exists in arachnids, and suggest that SsCl may be an in vivo target of ivermectin in S. scabiei.     

Field Releases of the Predatory Mite Neoseiulus fallacis (Acari: Phytoseiidae) in Canada, Monitored by Pyrethroid Resistance and Allozyme Markers

The predacious phytoseiid mite Neoseiulus fallacis (Garman) is an important agent for the biological control of spider mites in deciduous fruit orchards in North America and Canada. It would be helpful to monitor the fate of released individuals to improve the results of introductions of the predators in biological control trials. We have used two types of genetic markers, pyrethroid resistance and allozymes, for indirect estimation of the survival of N. fallacis introduced in an apple orchard in Ontario, Canada. Mite samples were submitted to toxicological tests. The polymorphism of four enzymes was studied in individual females using an isoelectric focusing technique. A mite sample was taken from the field, mass-reared in the laboratory, and selected for permethrin resistance. This strain was released on several apple trees treated with permethrin, and mite samples were collected from the same trees 10 to 90 days later. The genetic composition and the insecticide resistance level of this sample were compared to those of two other samples from trees where mites had not previously been released, either in the same orchard or in a neighboring block. A control susceptible strain was compared using mites collected earlier from trees on the same site but outside the present experiment. The mites collected from the release trees and those from the strain used for the releases were found to be genetically closely related, as judged from a small genetic distance, and from similar levels of insecticide resistance in both samples. The control samples from the nonrelease trees were genetically distant from these and displayed low resistance levels. These results indicate that the released genotypes established and persisted in the release trees for the period of the experiment. The utility of the two approaches in assessing the fate of released natural enemies is discussed.     

Toxicity and efficacy of selected pesticides and new acaricides to stored product mites (Acari: Acaridida)

Stored product mites can often infest stored products, but currently there is little information regarding the efficacy of pesticides that can be used for control. In this study we evaluated several common pesticides formulated from single active ingredients (a.i.) or commercially available mixtures (chlorpyrifos, deltamethrin, beta-cyfluthrin, and a combination of deltamethrin and S-bioallethrin), plus an acaricide composed of permethrin, pyriproxyfen and benzyl benzolate, for efficacy against Acarus siro, Tyrophagus putrescentiae, and Aleuroglyphus ovatus. The pesticides were incorporated into the mite diets in a dose range of 10–1000 μg a.i. g⁻¹ diet. Concentrations for suppression of 50 and 90% population growth and eradication (rC₀) of mites were fit to linear regression models. None of the tested pesticides gave complete eradication of A. siro, which was the most tolerant of the three mite species tested. The most effective pesticide Allergoff 175 CS was a combination product (a nano-capsule suspension of permethrin, pyriproxyfen and benzyl benzolate) labeled for dust mites, with rC₀ range of 463–2453 μg a.i. (permethrin) g⁻¹ diet depending on the species. Least effective were chlorpyrifos and deltamethrin.     

Mutations in the house fly Vssc1 sodium channel gene associated with super-kdr resistance abolish the pyrethroid sensitivity of Vssc1/tipE sodium channels expressed in Xenopus oocytes

The super-kdr insecticide resistance trait of the house fly confers resistance to pyrethroids and DDT by reducing the sensitivity of the fly nervous system. The super-kdr genetic locus is tightly linked to the Vssc1 gene, which encodes a voltage-sensitive sodium channel alpha subunit that is the principal site of pyrethroid action. DNA sequence analysis of Vssc1 alleles from several independent super-kdr fly strains identified two amino acid substitutions associated with the super-kdr trait: replacement of leucine at position 1014 with phenylalanine (L1014F), which has been shown to cause the kdr resistance trait in this species, and replacement of methionine at position 918 with threonine (M918T). We examined the functional significance of these mutations by expressing house fly sodium channels containing them in Xenopus laevis oocytes and by characterizing the biophysical properties and pyrethroid sensitivities of the expressed channels using two-electrode voltage clamp. House fly sodium channels that were specifically modified by site-directed mutagenesis to contain the M918T/L1014F double mutation gave reduced levels of sodium current expression in oocytes but otherwise exhibited functional properties similar to those of wildtype channels and channels containing the L1014F substitution. However, M918T/L1014F channels were completely insensitive to high concentrations of the pyrethroids cismethrin and cypermethrin. House fly sodium channels specifically modified to contain the M918T single mutation, which is not known to exist in nature except in association with the L1014F mutation, gave very small sodium currents in oocytes. Assays of these currents in the presence of high concentrations of cismethrin suggest that this mutation alone is sufficient to abolish the pyrethroid sensitivity of house fly sodium channels. These results define the functional significance of the Vssc1 mutations associated with the super-kdr trait of the house fly and are consistent with the hypothesis that the super-kdr trait arose by selection of a second-site mutation (M918T) that confers to flies possessing it even greater resistance than the kdr allele containing the L1014F mutation.     

Aerial dispersal of European red mites (Acari: Tetranychidae) in commercial apple orchards

Aerial dispersal of European red mite, Panonychus ulmi (Koch), in commercial apple orchards was estimated by trapping windborne mites. Studies were conducted at four orchards in eastern New York during 1989 and 1990 and at three orchards in western New York during 1989. In each orchard mites were trapped in three locations; the interior of the orchard, at the border of the orchard and in a field or woodlot beyond the orchard. Large numbers of mites were captured, even when the numbers of mites on apple foliage were well below levels where mite injury to leaves was visible (less than five per leaf). The log numbers of mites trapped were linearly related to the log density of mites on leaves and this relationship was consistent for each year and region the study was conducted. The trap captures among the three locations in and outside an orchard were highly correlated. The implications these findings may have on metapopulation dynamics and resistance to acaricide dynamics are discussed.     

Effect of a fluvalinate-resistance-associated sodium channel mutation from varroa mites on cockroach sodium channel sensitivity to fluvalinate, a pyrethroid insecticide

Fluvalinate is a pyrethroid insecticide that is widely used in the control of the varroa mite (Varroa destructor), an ecto-parasite of the honeybee. Previously we identified four fluvalinate-resistance-associated mutations in the sodium channel gene of the varroa mite. One of the mutations caused a leucine (L) to proline (P) change at 1770 in the linker connecting domains III and IV of the sodium channel. Interestingly, at the position corresponding to the L to P mutation, all known insect (including honeybee) sodium channel proteins already naturally contain a P residue (e.g., P1577 in the cockroach sodium channel BgNa(v)). To determine whether insect sodium channels are less sensitive to fluvalinate than arachnid sodium channels, we replaced P1577 with an L in a BgNa(v) variant (BgNa(v)1-1) and examined the sensitivity of the recombinant channel to fluvalinate. The P1577L substitution did not alter the gating properties of the BgNa(v)1-1 channel expressed in Xenopus oocytes. However, the BgNa(v)1-1(P1577L) channel was five-fold more sensitive to fluvalinate compared with the BgNa(v)1-1 channel. These results not only implicate the L to P mutation in fluvalinate resistance in varroa mites, but also suggest a possible contribution of L1770 to the higher sensitivity of varroa mites to fluvalinate than their insect hosts.     

The parasite-host specificity of the itch mite Sarcoptes scabiei (Acariformes: Sarcoptidae) in man and animals (a review of the literature)]

The primary parasitic relations between the itch mite Sarcoptes scabiei (L.) and man are confirmed indirectly by the presence in the family Sarcoptidae of close genera, members of which parasitize primates. Judging from the composition of domestic and wild animals-hosts, the source of their infection with S. scabiei was man that gave rise to numerous forms of mites specific to individual species of hosts or their related groups belonging to different taxonomic ranks. Three types of host relations and, accordingly, forms of sarcoptosis are suggested at the infection from an alien host: pseudosarcoptosis, temporary self-curable and typical lingering sarcoptosis. The ways of reinfection, invasiveness of stages of the mite and clinical picture for each form of the disease are analyzed.