Performance of CO1 and ITS2 nested PCR in molecular identification of ordinary scabies ( Sarcoptes scabiei var. hominis )

Scabies is a global disease with a high prevalence, causing morbidity and even mortality, especially in poor and developing countries. However, it is often misdiagnosed due to varied and unspecified lesions. The gold standard technique for diagnosis is a microscopic examination, which requires experienced experts in finding mites, mainly in ordinary scabies. CO1 and ITS2 genes have been widely used in molecular identification to detect Sarcoptes scabiei and its variants. This study aimed to determine and compare the sensitivity and specificity of CO1 and ITS2 S. scabiei genes to the microscopic examination of scabies skin scrapings. The skin scrapings of 52 subjects with scabies diagnosed by anamnesis, physical examination, and dermoscopic examination were examined under a microscope and analyzed by nested PCR. The diagnostic test result showed that the sensitivity of nested PCR of both CO1 and ITS2 genes to micro‐ scope examination was 100%. However, the specificity of both CO1 and ITS2 nested PCR was poor (24% and 0%). Hence, CO1 and ITS2 nested PCR could be more suitable for screening ordinary scabies in humans than the microscopic examination.


Introduction
Sarcoptes scabiei is an ectoparasite from the order Aca rina which causes scabies in humans and mammals. It has a high prevalence in developing countries with tropi cal climates and high humidity (Liu et al. 2016). Based on the 2015 Global Burden of Disease, Southeast Asia was ranked second with the highest prevalence of scabies af ter East Asia, and Indonesia was ranked first among 195 countries globally (Karimkhani et al. 2017a). Scabies is transmitted in crowded populations, close intrapersonal skin contact, lack of personal hygiene, low socioeconomic status, and unappropriated sexual behavior (Zayyid et al. 2010).
The most frequent scabies complication due to skin damage is bacterial infections. Streptococcus pyogenes and Staphylococcus aureus can lead to systemic infec tions such as acute poststreptococcal glomerulonephri tis (APSGN), rheumatic fever, and rheumatic heart dis ease. Consequently, scabies can cause morbidity based on disabilityadjusted life year (DALY) and even mortal ity (Karimkhani et al. 2017a,b). Scabies is also associated with intellectual disability (Liu et al. 2017b). Additionally, scabies has been linked to the production of IL17, a pro inflammatory cytokine, by the immune system. It is a sig nificant immunopathology factor in scabies patients suf fering from numerous autoimmune diseases, such as pso riasis and rheumatoid arthritis (Karimkhani et al. 2017a; Liu et al. 2017a).
The accuracy of scabies diagnosis is vital due to the high prevalence and magnitude of the impact of scabies. To date, the diagnosis is based on the presence of two of four cardinal signs as follows: nocturnal pruritus, living in crowded housing, typical lesions and predilections, and microscopic positive (found mites, eggs, or scibala). How ever, most patients present various and unspecified lesions that often cause misdiagnosis up to 65% (Hong et al. 2010; Sule andDankyau 2015). Furthermore, the difficulty of finding parasites during microscopic examination is due to fewer parasites, especially in ordinary scabies. ces left in skin lesions; a microscope cannot observe that. The result of this diagnostic tool can find genetic diver sity; even differences between varieties are limited. Mito chondrial haplotypes have been used to demonstrate that the genetic diversity of individual infestations is typically low, from just one to three distinct haplotypes per infes tation (Mofiz et al. 2016). The cytochrome c oxidase sub unit 1 (CO1), a gene in mitochondria of each organism's cell, will be amplified in large copy numbers and detected during PCR (Hahm et al. 2018). In addition, the CO1 gene is a potential marker since it can be distinguished from other human ectoparasite mites even with other variants of S. scabiei as a large amount of sequencing data exists (Park et al. 2012). The ITS2 gene, located in the mite ri bosome, is sensitive to detect S. scabiei DNA even after treatment. In addition, this gene can detect genetic varia tions of species that are related taxonomically (Fukuyama et al. 2010). The study aimed to verify the orientednested PCR of CO1 and ITS2 as a tool and screening marker of ordinary scabies.

Ethical statement
This study was approved by the ethical review board of the Faculty of Medicine, Sriwijaya University, Indonesia, in September 2018 (certificate number of 244/kepkrsmh fkunsri/2018). Written consent was obtained from each patient after informing the summary of the study.

Subjects of the study
This study used a diagnostic test design to assess the sen sitivity of the CO1 and ITS2 compared to the microscopic examination of scabies skin scrapings. The study was con ducted from October 2018 to April 2019. Subjects in this study were all students who lived in boarding schools, ful filled diagnostic criteria of scabies (cardinal signs), and provided informed consent. Consecutive sampling was conducted with a minimum of 47 students. Students pos itive for scabies by the presence of key symptoms were examined with a dermoscopy by a dermatologist to deter mine the location of mites. Only patients with two positive cardinal signs and dermoscopy of scabies were included in this study.
Skin scrapings and microscopic examinations were performed directly onsite at the subjects' residencies. The nested PCR analysis was conducted at the Biomolecular Laboratory of Medical Faculty, Sriwijaya University, In donesia.

Sample collection
Subjects with positive cardinal signs and dermoscopic ex amination were scrapped by scalpel no.15 and examined under a microscope with a magnitude of 100×. All sam ples were saved in an Eppendorf tube with alcohol absolute for storage.

DNA extraction and PCR amplification
According to the manufacturer's protocol, the DNA was extracted from the skin scrapings with the DNeasy Kit (Qiagen, Hilden, Germany). The nested PCR analysis was performed using an automatic thermocycler (Biorad) and Go Taq Green Hot Star (Promega, Madison, USA). The primers used are shown in Table 1. We used wob ble primers for the first round of CO1 amplification. New primers of secondround nested PCR were designed to im prove the sensitivity of the ITS2 gene.
PCR amplification was performed with 40 cycles of denaturation step at 95°C for 5 s, annealing temperature at 45°C (CO1) and 56.9°C (ITS2) for 5 s, 72°C for 20 s, and final extension 72°C for 1 min. Electrophoresis of the amplicon was carried out in Tris Borate EDTA (TBE) agarose gel 2% and ethidium bromide stain. The marker of visualization was DNA ladder 100 bp. It was confirmed to be positive when an amplicon of 317 bp for CO1 and 417 bp and 272 bp for the first and second round ITS2, respec tively. Three samples of each CO1 and ITS2 with clear target bands were then used for sequencing analysis. The sequencing was carried out at PT. Genetika Science and the Geneious program analyzed the result (Kearse et al. 2012).

Analysis
The nested PCR CO1 and ITS2 genes were analyzed using a 2×2 table for diagnostic test and Epicalc Statistical Soft ware (free to download at https://download.informer.co m/win11923940827270988d6e716f11/ec2v102.exe) to obtain sensitivity, specificity, positive predictive value, negative predictive value, and accuracy.

Result
In total, samples from 52 students were included in this study. The total sample was 53, but we excluded one sam ple, which showed confusing results of the second DNA extraction for amplification (no.16). The CO1 was not detected in the electrophoresis reading result, while ITS2 still did. The sociodemographic characteristics are pre sented in Table 2 and the distribution of subjects based on symptoms and the dermoscopic examination are pre sented in Table 3. The microscopic observation of S. sca biei mites and eggs are presented in Figure 1 and Figure  2. The positive results of a diagnostic test were compared  to the PCR test result and a microscopic examination to assess the clinical value of the nested PCR of the CO1 and ITS2 genes, as shown in Tables 4 and 5, respectively.
All samples identified as positive by microscopic ex amination also showed positive results by the nested PCR of CO1 and ITS2. These findings suggested that both genes produce 100% sensitivity. However, all samples identified as negative results by microscopic examination (34 samples) were identified as positive by nested PCR of CO1 gene (76.4%) and ITS2 (100%). These results mean that nested PCR of both genes produced poor specificity (Table 4 and 5). Electrophoresis reading results of CO1 and ITS2 genes are shown in Figure 3 and Figure 4.

Discussion
Risk factors of scabies transmission are environmental fac tors and personal hygiene. Most of the subjects included in this study were female students (73%). The humid and more closed conditions of the female dormitory may have contributed to scabies related to the lighting conditions, ventilation, humidity, and density of residents. The hu mid environment is directly proportional to the incidence of scabies (Liu et al. 2016). Mites and the eggs that can survive and are still infective outside the host are the en vironment's critical transmission (Hay et al. 2012; Arlian andMorgan 2017). Timely treatment and control at an early stage can prevent significant transmission, especially in potent areas like hospitals, jails and boarding schools, and in tropical and developing countries with a high preva lence of scabies and limited health care facilities (Park et al. 2012; Wong et al. 2015. A hospital outbreak that affected more than fifty patients and the staff was success fully cured after the community consisted of more than one thousand patients and caregivers in 3 months. It showed us that late diagnosis or screening of high prevalence scabies had a significant socioeconomic burden for patients and hospitals (Meyer et al. 2011). Furthermore, most of the subjects were new residents, accounting that 77% of suf   ferers had never had this disease before. Scabies transmis sion mainly manifests clinically in patients who have never been exposed before because of lack of memory immunity, with reinfestation only occurring in 40% of patients be cause protective immunity has been developed (Hay et al. 2012). The presence of two clinical symptoms out of the four cardinal signs is defined as a diagnostic cutoff. The noctur nal pruritus with the history of exact symptoms from other roommates was suffered up to 100%. Sleep disruption is a common symptom of scabies, with nocturnal itching being the most noticeable, as mite activity peaks at night. Mite secretion and excretion products are specific antigens that mediate rapid hypersensitivity reactions consisting of skin inflammation, rashes and itching. Mites secrete proteases that activate proteaseactivated receptor 2 (PAR2), an itch receptor. In addition, the immune response to mites also activates Th2, subsequently releasing several cytokines (including IL31) that cause itchiness. Postscabies itching can also occur due to immune response to antigen scabies and central nervous hypersensitivity (Jackson et al. 2007; Lavery et al. 2016; Ashok Nair et al. 2016. Most subjects had few atypical lesions, confirming previous research that scabies generally presents with more than one type of lesion. Also, pathognomonic le sions, a form of tunnels with papules at the ends, are in frequent (Jackson et al. 2007). The topography of the sca bies was mostly between the fingers, like a previous study (Hay et al. 2012). The dominant topography of this study was due to direct skin contact being more common via hands. In addition, this location has a thin and loose stra tum corneum, making mite penetration easier (Walter et al. 2011).
The gold standard of scabies diagnosis is the micro scope examination. The microscopic result of scabies in festation in our study is relatively high. Of the 52 skin scrapings samples, 18 (34.6%) contained mites and eggs of S. scabiei. A study in 2011 reported 46%, whereas an other study in 2015 that only 17 out of 100 patients were microscopically positive (Walter et al. 2011; Wong et al. 2015. Commonly, atypical lesions in place of mites or eggs caused this low sensitivity, although dermoscopic ex aminations were performed before. Dermoscopic obser vations can provide false positives, especially in hyperpig mented skin, excoriated and crusted skin. Also, scratching by the subject can cause mechanical removal of the mite; thus, the microscopic examination yields a false negative. Most subjects had superinfection that decreased sensitivity because pus in the lesion is acaricide (Walter et al. 2011).
The microscope method has a lower sensitivity, more over depends on the capability to scrape the skin and iden tify the mites, than a more sensitive test which is the biomolecular examination using PCR. Nested PCR analy sis of the skin scraping samples that had already been ex amined microscopically revealed that 18 microscopic pos itive skin scrapings were positive (100%) and 26 of 34 microscopic negative samples were positive (76.47%) for the CO1. A higher number than a previous study, which was14.4% and 25.7% using the same gene (Walter et al. 2011; Hahm et al. 2018. The sensitivity of nested PCR for CO1 genes to microscopic examination was 100%. The same sensitivity was also found in previous studies at 100%, which detected 28 positivePCR samples but only detected nine from 25 microscopic negative sam ples (Hahm et al. 2018). Regarding the ITS2 gene nested PCR, all samples (100%), both positive and negative mi croscopic skin scrapings, were analyzed. Previous stud ies were only able to identify 29 out of 53 positive micro scopic samples (Fukuyama et al. 2010). PCR as a screen ing tool could be valuable for scabies, as a community densely populated disease and communicable disease out break between passengers ship as reported by the Maritim Declaration of Health (MDH) (Hadjichristodoulou et al. 2011). This shows that the identification of nested PCR CO1 and ITS2 genes produced 100% sensitivity to the gold standard microscope examination.
The weakness of this current study is the low speci ficity of the PCR method for the diagnosis of scabies. The low specificity of PCR suggests for environment surveil lance from desquamated skin, dust, or fomites samples that could contain living mite or egg (Hay et al. 2012; Wong et al. 2015. A negative microscopic sample still posi tive on the PCR method produced a false negative because PCR still detects DNA part of scabies even though mite has died (Fukuyama et al. 2010; Hay et al. 2012. PCR can also be considered an additional analysis in cases where clini cal, dermoscopic or microscopic examination is negative, for example, in atypical and hidden scabies (Bezold et al. 2001).
We suggest further research on the immunodiagnostic of ordinary scabies, such as enzymelinked immunosor bent assay (ELISA) to detect scabiesspecific antibodies. In humans, this seems not feasible because of low var. hominis mite burden as antigen source and inability to take whole mite antigen (WMA) extract from in vitro cul ture. More than animals, recentstudy already use ELISA with WMA extract to detect mange and are commercially available for canis infestation. Some animal studies de velop recombinant immunogenic proteins such as Sar S 14.3, cofilin, and protein tyrosine kinase with up to 100% sensitivity and specificity scabies (Rampton et al. 2013). Wholegenome sequences of this potential diagnostic pro tein antigen are helpful to assess proteomic and genomic differences since scabies is known as the hostspecific agent. Its analysis also could minimize crossreactivity with house dust mites (Dean Rider et al. 2015). Some op timizations are worth considering the diagnostic tool of or dinary human scabies in the future.

Conclusions
In conclusion, dermoscopy orientednested PCR of CO1 and ITS2 could successfully identify S. scabiei infes tations, with ITS2 being more sensitive than CO1, so that it could be considered a potential molecular identi fication tool and screening marker of ordinary scabies. The sequence analysis identified several species varia tions in these genes (accession numbers KJ748523 and AB778919).