Novel miR-1958 Promotes Mycobacterium tuberculosis Survival in RAW264.7 Cells by Inhibiting Autophagy Via Atg5.

Autophagy is crucial for immune defense against Mycobacterium tuberculosis (Mtb) infection. Mtb can evade host immune attack and survival within macrophages by manipulating the autophagic process. MicroRNAs (miRNAs) are small, non-coding RNAs that are involved in regulating vital genes during Mtb infection. The precise role of miRNAs in autophagy with the exits of Mtb remains largely unknown. In this study, we found miR-1958, a new miRNA that could regulate autophagy by interacting with 3'UTR of autophagy-related gene 5 (Atg5). In addition, Mtb infection triggered miR-1958 expression in RAW264.7 cells. What's more, miR- 1958 overexpression blocked autophagic flux by impairing the fusion of autophagosomes and lysosomes. Overexpression of miR-1958 reduced Atg5 expression and LC3 puncta while inhibition of miR-1958 brought an increase of Atg5 and LC3 puncta; the opposite results were observed in detection of p62. The survival of Mtb in RAW264.7 cells transfected with mimic of miR-1958 was enhanced. Taken together, our research demonstrated that a novel miR-1958 could inhibit autophagy by interacting with Atg5 and favored intracellular Mtb survival in RAW264.7 cells.

The Construction of Full-Length cDNA Library for Otodectes cynotis.

BACKGROUND: Otodectes cynotis (Hering, 1838) is the pathogen of otodectic mange distributed worldwide. The mite mainly infests carnivores and, sometimes, humans. However, due to the lack of cDNA library, research on its pathogenesis has been challenging. METHODS: To solve this problem, the present study first sampled O. cynotis mites from an infested cat from Xi'an, China, for RNA extraction. Then, the full-length cDNA library was constructed using the SMART technique. Finally, positive clones > 500 bp and Hsc70-5 gene fragment specifically amplified from the cDNA library were sequenced and analyzed to verify the library's reliability. RESULTS: Results showed that RNA extracted from 300 mites had good quality with a concentration of 149 ng/µl and OD260/OD280 of 1.99. The library satisfied the quality standard of a good library with a titer of 5.02 × 105 PFU/ml and a combination rate of 97.61%. In addition, clone 4 and Hsc70-5 showed 98.38% and 99.72% identity with Ef1-α and Hsc70-5 gene sequences of O. cynotis in GenBank, respectively. CONCLUSION: The cDNA library of O. cynotis constructed here was successful and reliable, creating the basis for research on RNA sequencing and functional genes of O. cynotis.

Spatio-temporal pattern of schistosomiasis in Anhui Province, East China: Potential effect of the Yangtze River - Huaihe River Water Transfer Project.

Anhui Province has been one of typical epidemic areas of schistosomiasis in East China as a wide range of large lake and marshland regions provide an ideal environment for growth and reproduction of the intermediate snail host. With the completion of the Yangtze River-Huaihe River Water Transfer Project (YHWTP), launched by the end of 2016, the epidemic areas are expected to expand and controlling schistosomiasis remains a challenge. Based on annual surveillance data at the county level in Anhui for the period 2006-2015, spatial and temporal cluster analyses were conducted to assess the pattern of risk through spatial (Local Moran's I and flexible scan statistic) and space-time scan statistic (Kulldorff). It was found that schistosomiasis sero-prevalence was dramatically reduced and maintained at a low level. Cluster results showed that spatial extent of schistosomiasis contracted, but snail distribution remained geographically stable across the study area. Clusters, both for schistosomiasis and snail presence, were common along the Yangtze River. Considering the effect of the ongoing YHWTP on the potential spread of schistosomiasis, Zongyang County and Anqing, which will be transected by the new water-transfer route, should be given a priority for strengthened surveillance and control. Attention should also be paid to Guichi since it is close to one of the planned inlets of the YHWTP.

[Human Demodicosis: Emerging Dermatosis Caused by Demodex].

Demodex mites were discovered 175 years ago. However, the pathogenicity of Demodex remains questioned by clinicians due to the high infection yet low incidence of demodicosis and incomplete correlation between mite loads and clinical symptoms. In the recent decades, the pathogenicity of Demodex has attracted public concern as there are continuous case reports of skin lesions caused by mite infection. The pathogenicity of Demodex remains poorly understood. Demodex infection is suspected to be assocaited with rosacea, but its relation with other types of facial lesions such as papule and pustule is usually misdiagnosed or missed in clinical practice. This review gives an overview on the discovery, typical cases, pathogenesis, and clinical diagnosis and treatment of demodicosis, in the aim of promoting social recognition and attention on demodicosis.

Secondary structure of expansion segment D1 in LSU rDNA from Arachnida and its phylogenetic application in Eriophyoid mites and in Acari.

An increasing number of researchers have applied secondary-structure based multiple alignments of rDNA genes in phylogeny. These studies mostly depended on a few valuable divergent domains in LSU and SSU rDNA. Yet other divergent domains, e.g. D1, were poorly investigated and rarely used. However, these domains might contain additional evolutionary data and play a vital role in DNA-based phylogenetic study. Here, we investigated all available D1 sequences of Arachnida taxa and predicted corresponding secondary structures to help identify homologous positions in the D1 region. Long insertions were found exclusive to Eriophyoidea and folded into three newly proposed helices. Non-Acari taxa were all GC rich. In Acari, most Trombidiformes and all Mesostigmata (Parasitiformes) taxa were AT rich and Ixodida (Parasitiformes) GC rich; however there was no consistent base bias in Sarcoptiformes sequences. For Eriophyoid mites, genera Cecidophyopsis and Aceria were both well supported in MP, NJ, ME and ML tress based on D1 sequences, and clusters of Cecidophyopsis species were identical with former study. This demonstrated that the D1 region could act as a valuable molecular marker in phylogenetic reconstruction of Eriophyoidea. Additionally, D1 has been proven suitable in phylogenetic analysis at the family and genus level in Acari, but not in Opiliones.

Liver reserve function assessment by acoustic radiation force impulse imaging.

AIM: To evaluate the utility of liver reserve function by acoustic radiation force impulse (ARFI) imaging in patients with liver tumors. METHODS: Seventy-six patients with liver tumors were enrolled in this study. Serum biochemical indexes, such as aminotransferase (ALT), aspartate aminotransferase (AST), serum albumin (ALB), total bilirubin (T-Bil), and other indicators were observed. Liver stiffness (LS) was measured by ARFI imaging, measurements were repeated 10 times, and the average value of the results was taken as the final LS value. Indocyanine green (ICG) retention was performed, and ICG-K and ICG-R15 were recorded. Child-Pugh (CP) scores were carried out based on patient's preoperative biochemical tests and physical condition. Correlations among CP scores, ICG-R15, ICG-K and LS values were observed and analyzed using either the Pearson correlation coefficient or the Spearman rank correlation coefficient. Kruskal-Wallis test was used to compare LS values of CP scores, and the receiver-operator characteristic (ROC) curve was used to analyze liver reserve function assessment accuracy. RESULTS: LS in the ICG-R15 10%-20% group was significantly higher than in the ICG-R15 < 10% group; and the difference was statistically significant (2.19 ± 0.27 vs 1.59 ± 0.32, P < 0.01). LS in the ICG-R15 > 20% group was significantly higher than in the ICG-R15 < 10% group; and the difference was statistically significant (2.92 ± 0.29 vs 1.59 ± 0.32, P < 0.01). The LS value in patients with CP class A was lower than in patients with CP class B (1.57 ± 0.34 vs 1.86 ± 0.27, P < 0.05), while the LS value in patients with CP class B was lower than in patients with CP class C (1.86 ± 0.27 vs 2.47 ± 0.33, P < 0.01). LS was positively correlated with ICG-R15 (r = 0.617, P < 0.01) and CP score (r = 0.772, P < 0.01). Meanwhile, LS was negatively correlated with ICG-K (r = -0.673, P < 0.01). AST, ALT and T-Bil were positively correlated with LS, while ALB was negatively correlated with LS (P < 0.05). The ROC curve revealed that the when the LS value was 2.34 m/s, the Youden index was at its highest point, sensitivity was 69.2% and specificity was 92.1%. CONCLUSION: For patients with liver tumors, ARFI imaging is a useful tool for assessing liver reserve function.

[Research progress of taxonomy and discrimination techniques of Oncomelania hupensis].

Oncomelania hupensis, which plays an important role in the transmission of schistosomiasis japonica, is the only intermediate host of Schistosoma japonicum, and carrying out studies on its subspecies differentiation and genetic variation will have an important significance for schistosomiasis control. In this paper, the research progress of taxonomy and discrimination techniques of O. hupensis is reviewed at four levels, namely the morphological level, the cellular level, the protein level, and the DNA molecule level, and DNA sequencing technology is considered as the ideal taxonomy and discrimination technique of O. hupensis and the future research emphasis.

Molecular identification and phylogenetic study of Demodex caprae.

The DNA barcode has been widely used in species identification and phylogenetic analysis since 2003, but there have been no reports in Demodex. In this study, to obtain an appropriate DNA barcode for Demodex, molecular identification of Demodex caprae based on mitochondrial cox1 was conducted. Firstly, individual adults and eggs of D. caprae were obtained for genomic DNA (gDNA) extraction; Secondly, mitochondrial cox1 fragment was amplified, cloned, and sequenced; Thirdly, cox1 fragments of D. caprae were aligned with those of other Demodex retrieved from GenBank; Finally, the intra- and inter-specific divergences were computed and the phylogenetic trees were reconstructed to analyze phylogenetic relationship in Demodex. Results obtained from seven 429-bp fragments of D. caprae showed that sequence identities were above 99.1% among three adults and four eggs. The intraspecific divergences in D. caprae, Demodex folliculorum, Demodex brevis, and Demodex canis were 0.0-0.9, 0.5-0.9, 0.0-0.2, and 0.0-0.5%, respectively, while the interspecific divergences between D. caprae and D. folliculorum, D. canis, and D. brevis were 20.3-20.9, 21.8-23.0, and 25.0-25.3, respectively. The interspecific divergences were 10 times higher than intraspecific ones, indicating considerable barcoding gap. Furthermore, the phylogenetic trees showed that four Demodex species gathered separately, representing independent species; and Demodex folliculorum gathered with canine Demodex, D. caprae, and D. brevis in sequence. In conclusion, the selected 429-bp mitochondrial cox1 gene is an appropriate DNA barcode for molecular classification, identification, and phylogenetic analysis of Demodex. D. caprae is an independent species and D. folliculorum is closer to D. canis than to D. caprae or D. brevis.

Molecular identification of four phenotypes of human Demodex in China.

Traditional classification of Demodex mites by hosts and phenotypic characteristics is defective because of environmental influences. In this study, we proposed molecular identification of four phenotypes of two human Demodex species based on mitochondrial cox1 fragments for the first time. Mites collected from sufferers' facial skin were classified into four phenotypes: phenotype A-C with finger-like terminus, and phenotype D with cone-like terminus. The results of molecular data showed that cox1 sequences were all 429 bp. Divergences, genetic distances and transition/transversion ratios among the three phenotypes with finger-like terminus were 0.0-3.0%, 0.000-0.031, and 6/3-5/0, respectively, in line with intraspecific differences. However, those measures between the phenotype with cone-like terminus and phenotypes with finger-like terminus were 19.6-20.5%, 0.256-0.271, and 0.58 (31/53)-0.66 (35/53), respectively, reaching interspecific level. Phylogenetic trees also showed that the three phenotypes with finger-like terminus clustered as one clade, and the phenotype with cone-like terminus formed another one. Therefore, we conclude that mitochondrial cox1 sequence is a good marker for identification of two human Demodex species. Molecular data indicate no subspecies differentiation. Terminus is an effective character for species identification. Mites with finger-like terminus are Demodex folliculorum, and those with cone-like terminus are Demodex brevis.

Discrimination between Demodex folliculorum (Acari: Demodicidae) isolates from China and Spain based on mitochondrial cox1 sequences.

For a long time, classification of Demodex mites has been based mainly on their hosts and phenotypic characteristics. A new subspecies of Demodex folliculorum has been proposed, but not confirmed. Here, cox1 partial sequences of nine isolates of three Demodex species from two geographical sources (China and Spain) were studied to conduct molecular identification of D. folliculorum. Sequencing showed that the mitochondrial cox1 fragments of five D. folliculorum isolates from the facial skin of Chinese individuals were 429 bp long and that their sequence identity was 97.4%. The average sequence divergence was 1.24% among the five Chinese isolates, 0.94% between the two geographical isolate groups (China (5) and Spain (1)), and 2.15% between the two facial tissue sources (facial skin (6) and eyelids (1)). The genetic distance and rate of third-position nucleotide transition/transversion were 0.0125, 2.7 (3/1) among the five Chinese isolates, 0.0094, 3.1 (3/1) between the two geographical isolate groups, and 0.0217, 4.4 (3/1) between the two facial tissue sources. Phylogenetic trees showed that D. folliculorum from the two geographical isolate groups did not form sister clades, while those from different facial tissue sources did. According to the molecular characteristics, it appears that subspecies differentiation might not have occurred and that D. folliculorum isolates from the two geographical sources are of the same population. However, population differentiation might be occurring between isolates from facial skin and eyelids.

Phylogenetic analysis of Demodex caprae based on mitochondrial 16S rDNA sequence.

Demodex caprae infests the hair follicles and sebaceous glands of goats worldwide, which not only seriously impairs goat farming, but also causes a big economic loss. However, there are few reports on the DNA level of D. caprae. To reveal the taxonomic position of D. caprae within the genus Demodex, the present study conducted phylogenetic analysis of D. caprae based on mt16S rDNA sequence data. D. caprae adults and eggs were obtained from a skin nodule of the goat suffering demodicidosis. The mt16S rDNA sequences of individual mite were amplified using specific primers, and then cloned, sequenced, and aligned. The sequence divergence, genetic distance, and transition/transversion rate were computed, and the phylogenetic trees in Demodex were reconstructed. Results revealed the 339-bp partial sequences of six D. caprae isolates were obtained, and the sequence identity was 100% among isolates. The pairwise divergences between D. caprae and Demodex canis or Demodex folliculorum or Demodex brevis were 22.2-24.0%, 24.0-24.9%, and 22.9-23.2%, respectively. The corresponding average genetic distances were 2.840, 2.926, and 2.665, and the average transition/transversion rates were 0.70, 0.55, and 0.54, respectively. The divergences, genetic distances, and transition/transversion rates of D. caprae versus the other three species all reached interspecies level. The five phylogenetic trees all presented that D. caprae clustered with D. brevis first, and then with D. canis, D. folliculorum, and Demodex injai in sequence. In conclusion, D. caprae is an independent species, and it is closer to D. brevis than to D. canis, D. folliculorum, or D. injai.

Secondary structure prediction for complete rDNA sequences (18S, 5.8S, and 28S rDNA) of Demodex folliculorum, and comparison of divergent domains structures across Acari.

According to base pairing, the rRNA folds into corresponding secondary structures, which contain additional phylogenetic information. On the basis of sequencing for complete rDNA sequences (18S, ITS1, 5.8S, ITS2 and 28S rDNA) of Demodex, we predicted the secondary structure of the complete rDNA sequence (18S, 5.8S, and 28S rDNA) of Demodex folliculorum, which was in concordance with that of the main arthropod lineages in past studies. And together with the sequence data from GenBank, we also predicted the secondary structures of divergent domains in SSU rRNA of 51 species and in LSU rRNA of 43 species from four superfamilies in Acari (Cheyletoidea, Tetranychoidea, Analgoidea and Ixodoidea). The multiple alignment among the four superfamilies in Acari showed that, insertions from Tetranychoidea SSU rRNA formed two newly proposed helixes, and helix c3-2b of LSU rRNA was absent in Demodex (Cheyletoidea) taxa. Generally speaking, LSU rRNA presented more remarkable differences than SSU rRNA did, mainly in D2, D3, D5, D7a, D7b, D8 and D10.

Molecular identification of four phenotypes of human Demodex mites (Acari: Demodicidae) based on mitochondrial 16S rDNA.

Classification of Demodex mites has long depended on hosts and morphological characteristics. However, the fact that two species coexist in the same host and phenotype is easily influenced by environment causes difficulty and indeterminacy in traditional classification. Genotype, which directly reflects the molecular structure characteristics, is relatively stable. In this study, species identification of four phenotypes of human Demodex mites was conducted. Mites were morphologically classified into four phenotypes: long- and short-bodied Demodex folliculorum with finger-like terminus and Demodex brevis with finger- or cone-like terminus. The mitochondrial 16S ribosomal DNA (rDNA) fragment of individual mite was amplified, cloned, sequenced, and aligned. Sequence divergences, genetic distances, transition/transversion rates, and phylogenetic trees were analyzed. The results demonstrated that the 16S rDNA sequence of three phenotypes with finger-like terminus was 337 bp, and that of phenotype with cone-like terminus was 342 bp. The divergences, genetic distances, and transition/transversion rates among the three phenotypes with finger-like terminus were 0.0-2.7%, 0.000-0.029, and 5.0-7/0 (5/1-7/0), respectively, indicating an intraspecific variation. Yet, those between these three phenotypes and the one with cone-like terminus were 21.6-22.8%, 2.510-2.589, and 0.47-0.59 (22/47-27/46), respectively, suggesting an interspecific variation. The five phylogenetic trees showed that the three phenotypes with finger-like terminus clustered into one branch, while the phenotype with cone-like terminus clustered into another. In conclusion, terminus is a major morphological characteristic for the identification of human Demodex species. The three phenotypes with finger-like terminus belong to D. folliculorum, while the phenotype with cone-like terminus belongs to D. brevis. Molecular identification can verify and replenish morphological identification.

Cloning and sequence analysis of chitin synthase gene fragments of Demodex mites.

To our knowledge, few reports on Demodex studied at the molecular level are available at present. In this study our group, for the first time, cloned, sequenced and analyzed the chitin synthase (CHS) gene fragments of Demodex folliculorum, Demodex brevis, and Demodex canis (three isolates from each species) from Xi'an China, by designing specific primers based on the only partial sequence of the CHS gene of D. canis from Japan, retrieved from GenBank. Results show that amplification was successful only in three D. canis isolates and one D. brevis isolate out of the nine Demodex isolates. The obtained fragments were sequenced to be 339 bp for D. canis and 338 bp for D. brevis. The CHS gene sequence similarities between the three Xi'an D. canis isolates and one Japanese D. canis isolate ranged from 99.7% to 100.0%, and those between four D. canis isolates and one D. brevis isolate were 99.1%-99.4%. Phylogenetic trees based on maximum parsimony (MP) and maximum likelihood (ML) methods shared the same clusters, according with the traditional classification. Two open reading frames (ORFs) were identified in each CHS gene sequenced, and their corresponding amino acid sequences were located at the catalytic domain. The relatively conserved sequences could be deduced to be a CHS class A gene, which is associated with chitin synthesis in the integument of Demodex mites.

Sequencing for complete rDNA sequences (18S, ITS1, 5.8S, ITS2, and 28S rDNA) of Demodex and phylogenetic analysis of Acari based on 18S and 28S rDNA.

Due to the difficulty of DNA extraction for Demodex, few studies dealt with the identification and the phyletic evolution of Demodex at molecular level. In this study, we amplified, sequenced, and analyzed a complete (Demodex folliculorum) and an almost complete (D12 missing) (Demodex brevis) ribosomal DNA (rDNA) sequence and also analyzed the primary sequences of divergent domains in small-subunit ribosomal RNA (rRNA) of 51 species and in large-subunit rRNA of 43 species from four superfamilies in Acari (Cheyletoidea, Tetranychoidea, Analgoidea, and Ixodoidea). The results revealed that 18S rDNA sequence was relatively conserved in rDNA-coding regions and was not evolving as rapidly as 28S rDNA sequence. The evolutionary rates of transcribed spacer regions were much higher than those of the coding regions. The maximum parsimony trees of 18S and 28S rDNA appeared to be almost identical, consistent with their morphological classification. Based on the fact that the resolution capability of sequence length and the divergence of the 13 segments (D1-D6, D7a, D7b, and D8-D12) of 28S rDNA were stronger than that of the nine variable regions (V1-V9) of 18S rDNA, we were able to identify Demodex (Cheyletoidea) by the indels occurring in D2, D6, and D8.

Phylogenetic relationships in Demodex mites (Acari: Demodicidae) based on mitochondrial 16S rDNA partial sequences.

To confirm phylogenetic relationships in Demodex mites based on mitochondrial 16S rDNA partial sequences, mtDNA 16S partial sequences of ten isolates of three Demodex species from China were amplified, recombined, and sequenced and then analyzed with two Demodex folliculorum isolates from Spain. Lastly, genetic distance was computed, and phylogenetic tree was reconstructed. MEGA 4.0 analysis showed high sequence identity among 16S rDNA partial sequences of three Demodex species, which were 95.85 % in D. folliculorum, 98.53 % in Demodex canis, and 99.71 % in Demodex brevis. The divergence, genetic distance, and transition/transversions of the three Demodex species reached interspecies level, whereas there was no significant difference of the divergence (1.1 %), genetic distance (0.011), and transition/transversions (3/1) of the two geographic D. folliculorum isolates (Spain and China). Phylogenetic trees reveal that the three Demodex species formed three separate branches of one clade, where D. folliculorum and D. canis gathered first, and then gathered with D. brevis. The two Spain and five China D. folliculorum isolates did not form sister clades. In conclusion, 16S mtDNA are suitable for phylogenetic relationship analysis in low taxa (genus or species), but not for intraspecies determination of Demodex. The differentiation among the three Demodex species has reached interspecies level.

Complete sequence analysis of 18S rDNA based on genomic DNA extraction from individual Demodex mites (Acari: Demodicidae).

The study for the first time attempted to accomplish 18S ribosomal DNA (rDNA) complete sequence amplification and analysis for three Demodex species (Demodex folliculorum, Demodex brevis and Demodex canis) based on gDNA extraction from individual mites. The mites were treated by DNA Release Additive and Hot Start II DNA Polymerase so as to promote mite disruption and increase PCR specificity. Determination of D. folliculorum gDNA showed that the gDNA yield reached the highest at 1 mite, tending to descend with the increase of mite number. The individual mite gDNA was successfully used for 18S rDNA fragment (about 900 bp) amplification examination. The alignments of 18S rDNA complete sequences of individual mite samples and those of pooled mite samples ( ≥ 1000mites/sample) showed over 97% identities for each species, indicating that the gDNA extracted from a single individual mite was as satisfactory as that from pooled mites for PCR amplification. Further pairwise sequence analyses showed that average divergence, genetic distance, transition/transversion or phylogenetic tree could not effectively identify the three Demodex species, largely due to the differentiation in the D. canis isolates. It can be concluded that the individual Demodex mite gDNA can satisfy the molecular study of Demodex. 18S rDNA complete sequence is suitable for interfamily identification in Cheyletoidea, but whether it is suitable for intrafamily identification cannot be confirmed until the ascertainment of the types of Demodex mites parasitizing in dogs.

A meta-analysis of association between acne vulgaris and Demodex infestation.

Until now, etiology of acne vulgaris is still uncertain. Although clinicians usually deny the association between Demodex infestation and acne vulgaris, it has been proved in some clinical practices. To confirm the association between Demodex infestation and acne vulgaris, a meta-analysis was conducted. Predefined selection criteria were applied to search all published papers that analyzed the association between Demodex infestation and acne vulgaris (January 1950 to August 2011) in ISI Web of Knowledge, MEDLINE, and China National Knowledge Infrastructure (CNKI) databases. A meta-analysis was performed to calculate odds ratios (ORs) and 95% confidence intervals (CIs) based on fixed effects models or random effects models. We enrolled the 60 Chinese and 3 English papers in this meta-analysis, which covered Turkey and 25 different provinces/municipalities in China and 42130 participants including students and residents, aged from 1 to 78 years. The pooled OR in random effects models is 2.80 (95% CI, 2.34-3.36). Stability is robust according to sensitivity analysis. The fail-safe number is 18477, suggesting that at least 18477 articles with negative conclusions would be needed to reverse the conclusion that acne vulgaris was related to Demodex infestation. So the effect of publication bias was insignificant and could be ignored. It was concluded that acne vulgaris is associated with Demodex infestation. This indicates that when regular treatments for acne vulgaris are ineffective, examination of Demodex mites and necessary acaricidal therapies should be considered.

Association of blepharitis with Demodex: a meta-analysis.

PURPOSE: To conduct a meta-analysis to confirm the association between Demodex infestation and blepharitis. METHODS: We conducted a comprehensive and quantitative analysis of relevant published case-control studies which were found from the ISI Web of Knowledge, MEDLINE and CNKI from January 1950 to December 2010. Meta-analysis was applied for 13 of these and included matched data sets, using odds ratio (OR) as the effect indicator. Sensitivity was assessed. RESULTS: Eleven articles (13 matched data sets) covering four different countries and reporting 4741 participants (2098 blepharitis and 2643 controls) were eligible. The pooled OR in random effect models was 4.89 (95% confidence interval, 3.00-7.97). Sensitivity analysis showed that results of pooled ORs in different effect models, language, sample size, and control groups were completely consistent, which demonstrated a stable association between Demodex infestation and blepharitis by meta-analysis. CONCLUSIONS: The association between Demodex infestation and blepharitis was statistically significant. The conclusion implies that when conventional treatments for blepharitis fail, examination of Demodex mites and acaricidal therapy should be considered.

RAPD-SCAR marker and genetic relationship analysis of three Demodex species (Acari: Demodicidae).

For a long time, classification of Demodex mites has been mainly based on their hosts and phenotype characteristics. The study was the first to conduct molecular identification and genetic relationship analysis for six isolates of three Demodex species by random amplified polymorphic DNA (RAPD) and sequence-characterized amplified region (SCAR) marker. Totally, 239 DNA fragments were amplified from six Demodex isolates with 10 random primers in RAPD, of which 165 were polymorphic. Using a single primer, at least five fragments and at most 40 in the six isolates were amplified, whereas within a single isolate, a range of 35-49 fragments were amplified. DNA fingerprints of primers CZ 1-9 revealed intra- and interspecies difference in six Demodex isolates, whereas primer CZ 10 only revealed interspecies difference. The genetic distance and dendrogram showed the intraspecific genetic distances were closer than the interspecific genetic distances. The interspecific genetic distances of Demodex folliculorum and Demodex canis (0.7931-0.8140) were shorter than that of Demodex brevis and D. canis (0.8182-0.8987). The RAPD-SCAR marker displayed primer CZ 10 could be applied to identify the three Demodex species. The 479-bp fragment was specific for D. brevis, and the 261-bp fragment was specific for D. canis. The conclusion was that the RAPD-SCAR multi-marker was effective in molecular identification of three Demodex species. The genetic relationship between D. folliculorum and D. canis was nearer than that between D. folliculorum and D. brevis.