Data collection

This study was conducted at the Tianjin Academy of Traditional Chinese Medicine Affiliated Hospital, in Tianjin, China. Tianjin is the second largest city in northern China. Most of the subjects in this study come from Tianjin, with a small portion coming from other regions of China. Microscopic images and the associated results from subjects who underwent optical microscopy examination of skin scrapings for the diagnosis of scabies between 2018 and 2022 were retrieved. This study did not require personal information from patients. It was approved and monitored by the hospital ethics committee, which approved an exemption from obtaining patient consent.

Subjects

The subjects who underwent optical microscopy examination were selected by clinicians based on clinical evaluation, including the following individuals:

1. (1) Individuals with confirmed scabies, who required an efficacy evaluation after treatment;

2. (2) Individuals with clinical or suspected scabies, who required a diagnosis of confirmed scabies;

3. (3) Individuals with other skin diseases, who required a differential diagnosis from scabies;

4. (4) Individuals with a history of contact with other individuals with confirmed scabies, who required to determine whether they were infected with scabies.

Morphological classification method of multiple diagnostic features of S. scabiei

The diagnostic features of S. scabiei observed under an optical microscope were classified into 5 categories and 7 subcategories.

1. (I) Mites. The developmental stages of S. scabiei include eggs, larvae, nymphs (protonymphs and tritonymphs) and adults (Lydeamore et al., Reference Lydeamore, Campbell, Regan, Tong, Andrews, Steer, Romani, Kaldor, McVernon and McCaw2019). In this study, mites refer to immature (larvae and nymphs) and mature (adult) forms (Fig. 1A–E). The anatomical structures of a mite include a gnathosoma and an idiosoma; the idiosoma bears setae, cuticular spines, cuticular striations, long bristles and 3 (larvae) or 4 (nymphs and adults) pairs of legs (Nonaka et al., Reference Nonaka, Abe and Sasai1985; Walton and Currie, Reference Walton and Currie2007; Nelson et al., Reference Nelson, Song and Ng2019) (Fig. 1A–E). Most of the anatomical structures of a mite should be complete. In some cases, a mite can be cut into several parts by a sterile blade in the process of extracting materials; however, the gnathosoma and most idiosoma structures should be observed in the same field under an optical microscope (Fig. 1F). Note that the eggshells of larvae should be either absent or incomplete (Fig. 1G–J). Examples and the anatomical structures are shown in Fig. 1.

2. (II) Eggs. Eggs refer to ova or eggs with complete eggshells. A complete eggshell is the key characteristic of this category (Fig. 2).

3. (III) Mite fragments. Mite fragments are incomplete structures of mites cut using a sterile blade (Fig. 3). This category includes 3 subcategories: gnathosoma-type, idiosoma-type and leg-type fragments. Gnathosoma-type fragments refer to incomplete structures with gnathosoma (Fig. 3 A1–A4); whereas idiosoma-type fragments refer to incomplete structures without gnathosoma (Fig. 3 B1–B4); and leg-type structures are incomplete structures with only legs (Fig. 3 C1–C4). In contrast to mites, the gnathosoma and most body structures of idiosoma should not be observed in the same field under an optical microscope.

4. (IV) Eggshell fragments. Eggshell fragments are incomplete eggshells left by eggs after hatching into larvae. As shown in Fig. 4, the eggshell fragments exhibited different shapes. Based on the differences in size and shape, this category can be classified into 4 subcategories: double-leaf, single-leaf, 1-shaped and amorphous fragments.

Figure 1. Examples of mites and the anatomic structures. (A) Larvae; (B) nymphs; (C) adult male mites; (D) adult female mites (ventral aspect); (E) adult female mites (dorsal aspect); (F) a mite was cut into 2 parts; however, the gnathosoma and most idiosoma structures can be observed in a field; （G-J） larva with incomplete eggshells (red arrow). Complete body structures: a, gnathosoma; b-d, legs Ⅰ, Ⅱ, Ⅲ, and Ⅳ; f, long bristles; g, cuticular striations; h, cuticular spines; i, setae; j, ovum (magnification: 100).

Figure 2. Eggs at different developmental levels (magnification: 100).

Figure 3. Mite fragments. (A1–A4) Gnathosoma-type fragments; (B1–B4) idiosoma-type fragments; (C1–C4) leg-type fragments (magnification: 100).

Figure 4. Eggshell fragments. (A1–A4) Double-leaf fragments; (B1–B4) single-leaf fragments; (C1–C4) 1-shaped fragments; (D1–D4) amorphous fragments (magnification: 100).

Double-leaf fragments are relatively large. In some cases, the eggshell is slightly damaged with only 1 chink (Fig. 4 A1); in most cases, the eggshell is broken into 2 connected fragments, shaped like the letter ‘V’ (Fig. 4 A2–A4).

Single-leaf fragments are smaller than the double-leaf fragments. The eggshell is broken into 2 unconnected fragments shaped like a boat, helmet or pan (Fig. 4 B1–B4).

If an eggshell is severely damaged, several small fragments will remain. Some small fragments are shaped like the Arabic numeral ‘1’, which are referred to as 1-shaped fragments (Fig. 4 C1–C4); some irregular small fragments are referred to as amorphous fragments (Fig. 4 D1–D4). Note that the 1-shaped and amorphous fragments are easily confused with contaminating artefacts.

1. (A) Fecal pellets (scybala). Fecal pellets are the products of mites and are seen as small, oval structures that are easily confused with contaminating artefacts. Therefore, fecal pellets must appear in piles (Fig. 5).

Figure 5. Fecal pellets. (A and B) Fecal pellets in piles; (C) fecal pellets and a double-leaf fragment; (D) fecal pellets and a mite (magnification: 100).

Optical microscopy examination

The examination was performed by operators with more than 3 years of clinical experience as described elsewhere (Engelman et al., Reference Engelman, Yoshizumi, Hay, Osti, Micali, Norton, Walton, Boralevi, Bernigaud, Bowen, Chang, Chosidow, Estrada-Chavez, Feldmeier, Ishii, Lacarrubba, Mahé, Maurer, Mahdi, Murdoch, Pariser, Nair, Rehmus, Romani, Tilakaratne, Tuicakau, Walker, Wanat, Whitfeld, Yotsu, Steer and Fuller2020). Firstly, several suspected sites were determined based on multiple skin lesion morphologies involving finger web-spaces, hands, the volar surfaces of the wrists, axillae, buttocks, the areola in women, the toe seams in infants or young children and genitalia in men. Following that, skin scrapings were collected from the suspected sites on the same individual using a sterile blade (#15) after disinfection with 75% alcohol. The scrapings were then carefully placed on a standard glass microscope slide, mixed with 1–2 drops of potassium hydroxide (KOH) at a 15% concentration, and covered with a coverslip. The slide was left to stand for several minutes before being scanned under a microscope (Olympus CX33; Tokyo, Japan) at 100× magnification to search for the diagnostic features of S. scabiei. Finally, a field was selected according to the priority order of diagnostic features stated as follows, and then saved as a microscopic image using a camera (JEDA SmartVF650DO, Jiangsu, China) along with its automated imaging system.

Priority order of diagnostic features of S. scabiei

Some diagnostic features (mites and eggs) are easy to identify, whereas others (mite and eggshell fragments, and fecal pellets) may be difficult to identify or easily confused with contaminating artefacts. Meanwhile, multiple diagnostic features can be observed in a single examination. In this study, the priority order of diagnostic features is as follows: (1) mites; (2) eggs; (3) double-leaf fragments; (4) leg-type fragments; (5) single-leaf fragments; (6) gnathosoma-type fragments; (7) idiosoma-type fragments; (8) 1-shaped fragments; (9) amorphous fragments; (10) fecal pellets.

Positive detection rates

In this study, a positive case refers to an optical microscopy examination in which at least 1 diagnostic feature of S. scabiei was observed, whereas a negative case refers to an optical microscopy examination in which no diagnostic features of S. scabiei were observed. The annual and overall numbers of positive cases and total cases between 2018 and 2022 were counted. The annual and overall positive detection rates were then calculated as shown in equation (1).

(1)$$Positive\;detection\;rate = \displaystyle{{Positive\;cases} \over {Total\;cases}} \times 100\% $$

Frequency distribution of various diagnostic features of S. scabiei

According to the priority order of diagnostic features, all positive cases between 2018 and 2022 were divided into 10 groups: mites, eggs, double-leaf fragments, leg-type fragments, single-leaf fragments, gnathosoma-type fragments, idiosoma-type fragments, 1-shaped fragments, amorphous fragments, and fecal pellets. The numbers of positive cases in each group n_i (i = 1, …, 10) and the total positive cases were counted. The frequency of each group was then calculated as shown in equation (2).

(2)$$Frequency( i ) = \displaystyle{{n_i} \over {Total\;positive\;cases}} \times 100\% $$

Statistical analysis

All statistical analyses were performed using Microsoft Excel. Positive detection rates and frequencies were used for descriptive analysis, and the statistical results of the descriptive studies were directly compared.