• Users Online: 1066
  • Home
  • Print this page
  • Email this page
Home About us Editorial board Ahead of print Current issue Search Archives Submit article Instructions Subscribe Contacts Login 


 
 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 15  |  Issue : 3  |  Page : 432-437

Conventional and molecular diagnosis of clostridium difficile infections in a tertiary care hospital


1 Department of Microbiology, KIMSDU, Karad, Maharashtra, India
2 Department of Molecular Biology and Genetics, KIMSDU, Karad, Maharashtra, India
3 Department of Medicine, KIMSDU, Karad, Maharashtra, India

Date of Submission17-Aug-2020
Date of Decision20-Sep-2020
Date of Acceptance25-Sep-2020
Date of Web Publication1-Feb-2021

Correspondence Address:
Dr. Priyanka M Mane
Department of Microbiology, KIMSDU, Karad, Maharashtra
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jdmimsu.jdmimsu_163_19

Rights and Permissions
  Abstract 


Introduction: Indiscriminate use of broad-spectrum antibiotics has dramatically increased the incidence of Clostridium difficile-associated diarrhea (CDAD) in recent years. It is the most common cause of antibiotic-associated diarrhea (AAD) responsible for one-third of AAD cases. Aim: The aim was to study C. difficile in AAD. Objectives: The objectives were to study the prevalence of CDAD, to isolate C. difficile from AAD, and to study the molecular detection of toxin-producing strains of C. difficile. Materials and Methods: A total of 222 patients of AAD were assessed for C. difficile over a period of 2 years. Anaerobic culture for C. difficile was done on cycloserine-cefoxitin-fructose agar and brain–heart infusion agar. Enzyme-linked immunosorbent assay (ELISA) for toxin A and toxin B was used for detecting toxigenic strains of C. difficile. Identification of C. difficile and toxin-producing strains was done with the help of polymerase chain reaction (PCR). Observation and Results: Out of the total 222 cases of AAD, 20 (9%) were positive by culture and 70 (31.53%) were found to be toxin-producing C. difficile by ELISA. C. difficile was positive by PCR in 32 (14.41%); of these, 18 (56.25%) isolates were toxigenic, i.e., they possessed either the tcdA or the tcdB gene or both. Among the toxigenic isolates, 10 (31.25%) possessed both of the toxigenic genes (tcdA and tcdB) and the remaining 8 (25%) had one of the toxin genes. Only the toxin A (tcdA+ tcdB-) gene was found in 4 (12.5%) and only the toxin B (tcdA- tcdB+) gene in 4 (12.5%) of the toxigenic isolates. Conclusion: CDAD is an emerging nosocomial infection. Frequent and indiscriminate use of antibiotics has increased the prevalence of C. difficile infection. Conventional and molecular diagnosis can help to accurate diagnosis of these infections.

Keywords: Antibiotic-associated diarrhea, Clostridium difficile, Clostridium difficile-associated diarrhea


How to cite this article:
Mane PM, Patil SR, Datkhile KD, Mane MB, Karande GS. Conventional and molecular diagnosis of clostridium difficile infections in a tertiary care hospital. J Datta Meghe Inst Med Sci Univ 2020;15:432-7

How to cite this URL:
Mane PM, Patil SR, Datkhile KD, Mane MB, Karande GS. Conventional and molecular diagnosis of clostridium difficile infections in a tertiary care hospital. J Datta Meghe Inst Med Sci Univ [serial online] 2020 [cited 2021 Feb 28];15:432-7. Available from: http://www.journaldmims.com/text.asp?2020/15/3/432/308550




  Introduction Top


Antibiotic-associated diarrhea (AAD) is defined as otherwise unexplained diarrhea that occurs in association with the administration of antibiotics. The frequency of this complication varies among the antibacterial agents. Indiscriminate use of broad-spectrum antibiotics has dramatically increased the incidence of Clostridium difficile-associated diarrhea (CDAD) in recent years.[1] It is the most common cause of AAD responsible for one-third of AAD cases, 50%–75% of antibiotic-associated colitis, and 90%–100% cases of pseudomembranous colitis.[1],[2],[3] Toxin A and toxin B are the major virulence factors of C. difficile contributing to its pathogenicity.[4]

In India, the studies on C. difficile AAD are limited. The incidence of CDAD in hospitalized patients with diarrhea is estimated to be around 7.1%–30% in different Indian studies.[5],[6],[7],[8]

The dramatic change in the epidemiology of C. difficile infection during recent years, in both frequency and severity, owing to the emergence of virulent strains such as NAP1/BI/027 (North American Pulsed Field type 1/Restriction Endonuclease Assay type BI/Ribotype 027) in North America,[9] ribotype 078 in Europe,[10] and ribotype 017 in Asia,[11] has made C. difficile a public health concern. This strain was found to produce >16 times toxin A and 23 times toxin B in addition to the binary toxin.[12] Thus, considering C. difficile as an important nosocomial pathogen, the study was undertaken to find the prevalence of C. difficile in AAD, isolation of C. difficile, and detection of toxin-producing strains.


  Materials and Methods Top


The prospective study was conducted in the Department of Microbiology, Krishna Hospital and Medical Research Centre, a tertiary care hospital attached to medical college, at Krishna Institute of Medical Sciences Deemed to be University (KIMSDU), Karad, Western Maharashtra. The study was approved by the Ethical Committee of KIMSDU, Karad, Maharashtra. Written informed consent was taken from all the patients or their wards.

Study population

A total of 222 patients of AAD were assessed for C. difficile, from February 2017 to March 2019. Patients admitted in the hospital for ailments other than diarrhea and developed diarrhea after 72 h of antibiotic administration above the age of 2 years were included in the study. Patients admitted in the hospital for diarrhea due to other reasons were excluded from the study. Data were collected in pretested pro forma specially designed for this purpose which included identification, demographic, and clinical data.

Stool culture

All the stool specimens were processed immediately for culture of C. difficile. Culture for C. difficile was done on cycloserine-cefoxitin-fructose agar and brain–heart infusion agar.[20] Concurrently, a loopful of stool specimen was inoculated into Robertson's cooked meat broth and incubated at 37°C for 48 h. The plates were incubated anaerobically at 37°C in an anaerobic jar for 48 h. After incubation, the plates were examined and colonies which resembled C. difficile were identified by standard methods.[13]

Detection of enterotoxin and cytotoxin by enzyme-linked immunosorbent assay

Detection of enterotoxin and cytotoxin (toxin A and toxin B) of C. difficile was performed on the stool specimen according to manufacturer's instructions by a double-sandwich enzyme-linked immunosorbent assay (ELISA) technique using a commercial kit (Premier® Toxins A and B, Meridian Bioscience, Europe).

Identification of Clostridium difficile in Stool samples with the help of polymerase chain reaction

Stool specimens were subjected to DNA extraction. DNA was extracted using commercial kit (Qiagen kit – DNA extraction on stool sample) according to manufacturer's instructions.

For molecular investigation of C. difficile-specific genes and toxins, C. difficile ATCC 43255 strain was used as a positive control. The polymerase chain reaction (PCR) amplification and optimal annealing temperatures of the PCR primers of individual genes were initially optimized with gradient PCR in a Mastercycler Gradient (Eppendorf India Ltd.). All PCRs were performed in a volume of 20 μl, using 0.2 ml tubes with flat caps (Axygen). The amplified products after a gradient PCR were subjected to electrophoresis and checked for the single expected band and also were compared with 100 base pair ladder. The cde gene primers were used for detection and confirmation of the presence of C. difficile in the clinical stool samples. The virulence genes of C. difficile from the clinical stool samples were identified by PCR amplification using tcdA and tcdB gene primers. The primers used for the amplification of different genes of C. difficile are represented in [Table 1].
Table 1: Oligonucleotides used for the study

Click here to view


All PCR reactions were performed in a Mastercycler Gradient (Eppendorf, India) in 20 μl reaction mixtures containing 1 U Taq DNA polymerase (GeNei, India), 1 Xassay buffer consisting of 10 mM Tris-HCL (pH 9.0 at 2.5 0 C) 1.5 mM MgCl2, 50 mM KCL and 0.01% gelatin, each dNTP at a concentration of 200 μM, 1 Unit of Taq DNA polymerase enzyme and 10 pmoles of each oligonucleotides primers per reaction as mentioned in [Table 2] and water to make up to the final volume.
Table 2: Polymerase chain reaction programs used for amplification of toxin genes

Click here to view


The thermal cycling conditions started with an initial denaturation step at 95°C for 10 min followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 45°C–55°C for 30 s, an extension at 72°C for 60 s, and final extension 72°C for 10 min. After confirmation of annealing temperature conditions for all genes separately, the PCR amplification of cde, tcdA, and tcdB genes was carried out using the PCR programs, as mentioned in [Table 2].[14],[15],[16]

Statistical analysis

Summarization and analysis of data were carried out using the software Statistical Package for the Social Sciences (version 21) (SPSS Statistics for Windows, version x.0, SPSS Inc., Chicago, Ill., USA). Data were condensed in the form of tables. Data were also presented in the form of graphs/diagrams. Statistics such as percentages and mean were computed. The Chi-square test was said to be significant when probability was <0.05.

Ethical clearance

The Institutional Ethics Committee of KIMSDU has approved the Research work proposed to be carried out at KIMSDU, Karad, Maharashtra Date: 7th Oct 2016 with Reference no KIMSDU/EC/2016/648.


  Observation and Results Top


A total of 222 cases of AAD were included in the study. The study was conducted from February 2017 to March 2019. Stool samples were collected from the patients admitted in Krishna Hospital and Medical Research Centre for reason other than diarrhea and developed diarrhea after 72 h of antibiotic administration.

Out of the total 222 cases of AAD, 138 (62.16%) were male and 84 (37.83%) were female. The incidence of AAD was higher in females below 30 years of age; however, it was higher in males after 40 years of age. The difference was statistically significant (?2 = 20.59; df = 6; P < 0.05).

[Table 3] shows the ward-wise distribution of AAD cases and CDAD cases. A maximum number of AAD cases were from medical intensive care unit (MICU) (91, 40.99%), followed by medicine ward (41, 18.46%) and surgical intensive care unit (28, 12.61%), respectively.
Table 3: Ward.wise distribution of antibiotic.associated diarrhea cases and Clostridium difficile-associated diarrhea cases

Click here to view


Maximum cases were positive by ELISA for C. difficile, i.e., 32 (14.41%), and were from MICU, followed by medicine ward (11, 4.95%). Maximum culture-positive cases were from MICU (8, 3.6%), followed by medicine ward, i.e., 5 (2.3%). A maximum number of CDAD cases by PCR were from MICU (14, 6.3%).

[Table 4] shows the categorization of antibiotics causing AAD and ELISA, culture, and PCR positivity, respectively. Third-generation cephalosporins were the major group of antibiotic responsible for 69 (31.1%) and 21 (9.5%) cases of AAD and 7 (3.2%) and 14 (6.3%) cases of CDAD as detected by ELISA, culture, and PCR, respectively.
Table 4: Categorization of antibiotics causing antibiotic-associated diarrhea and Clostridium difficile-associated diarrhea

Click here to view


All the stool samples of AAD were processed according to the standard laboratory procedures. Each stool sample was subjected to culture, ELISA for toxins of C. difficile, and PCR. Out of the total 222 cases of AAD, 20 (9%) were positive by culture, 70 (31.53%) were found to be toxin-producing C. difficile by ELISA, and 32 (14.4%) were positive for cde gene (species-specific gene of C. difficile) by PCR. Overall, 74 cases gave evidence of C. difficile infection either by culture, ELISA, or PCR. Thus, in the present study, the prevalence of C. difficile infection was 33.33%.

Total 18 (8.1%) cases were positive by culture, ELISA, and PCR. Only 2 (0.9%) culture-positive cases were also positive by PCR but negative by ELISA. Total 42 (18.9%) cases were positive only by ELISA. Twenty-eight (12.6%) cases were positive for both ELISA and PCR but culture negative. Only 2 (0.9%) cases were positive by PCR alone. Total 148 (66.67%) cases did not show evidence of C. difficile infection [Table 5].
Table 5: Comparison of culture ELISA and polymerase chain reaction

Click here to view


[Table 6] shows the sensitivity and specificity of culture and ELISA. Considering cde detection of gene by PCR as a gold standard, the sensitivity of culture was calculated as 62.5% and specificity 100%. The same way sensitivity of ELISA was 87.5% and specificity 77.9%.
Table 6: Sensitivity and specificity of the tests

Click here to view


Identification of genes of Clostridium difficile

Evaluation of C. difficile for cde, tcdA, and tcdB genes was done by PCR.

C. difficile was positive for the species-specific cde gene identified by PCR in 32 (14.41%) of total 222 stool samples. Of these, 18 (56.25%) isolates were toxigenic, i.e., they possessed either the tcdA or the tcdB gene or both. Among the toxigenic isolates, 10 (31.25%) possessed both of the toxigenic genes (tcdA and tcdB) and the remaining 8 (25%) had one of the toxin genes. Only the toxin A (tcdA+ tcdB-) gene was found in 4 (12.5%) and only the toxin B (tcdA- tcdB+) gene in 4 (12.5%) of the toxigenic isolates [Table 7].
Table 7: Distribution of cde and toxin (tox A and tox B) producing strains

Click here to view



  Discussion Top


Worldwide, the incidence of C. difficile-associated infections has increased in recent years and has contributed to significant morbidity and mortality. C. difficile is now recognized as the primary cause of hospital-acquired colitis in patients who receive antibiotics.[17]

Overall, 74 cases gave evidence of C. difficile infection either by culture, ELISA, or PCR.

Our study reported the prevalence of 33.33% which is higher than the prevalence shown in other Indian studies.[5],[6],[7],[8] The overall prevalence in our study was high, which may be due to more number of toxin-positive cases detected by ELISA (70, 31.53%). Considering cde detection of gene by PCR as a gold standard, we found that the sensitivity of ELISA was 87.5% and specificity 77.9%.

In the present study, the incidence of AAD was higher in females below 30 years of age; however, it was higher in males after 40 years of age. The difference was statistically significant (?2 = 20.59; df = 6; P < 0.05). The mean age of the study participants was 40.66 years, standard deviation ± 16.969. In a study by Segar et al.,[18] the mean age of the study patients was 45.93 ± 16.65 (range, 5–82). Of the 150 AAD patients, 89 (59.3%) were male and 61 (40.7%) were female.

A conventional method like anaerobic culture was used for isolation of C difficile. The disadvantage of culture is that it lacks specificity. Culture even detects the nontoxigenic strains. In this study, we used culture and PCR for diagnosing C. difficile infection. ELISA and PCR were used for detecting the toxigenic isolates. Total 18 (8.1%) cases were positive by culture, ELISA, and PCR. Only 2 (0.9%) culture-positive cases were also positive by PCR but negative by ELISA. Total 42 (18.9%) cases were positive only by ELISA. Twenty-eight (12.6%) cases were positive for both ELISA and PCR but culture negative. Only 2 (0.9%) cases were positive by PCR alone [Figure 1] and [Figure 2].
Figure 1: Representative agarose gel image showing polymerase chain reaction amplification of 157bp fragment of cde gene of Clostridium difficile; Lane-1: 100 bp DNA marker; Lane-2: Positive control, Lane-3 to 10: Sample screened for cde gene where samples 166 and 170 showed presence of cde gene. Lane-11: Negative control

Click here to view
Figure 2: Representative agarose gel image showing polymerase chain reaction amplification of (a) 624 bp fragment of tcdA gene of Clostridium difficile; Lane-1; 100 bp DNA marker; Lane-2: Positive control, Lane-3: Sample 89, Lame-4: Sample 95, Lane-5: Sample 108. Lame-6: Negative control where samples 89 and 95, Lane-4: Sample 95, Lane-5: Sample 108. Lane-6: Negative control where samples 89 and 95 showed the presence of tcdA gene. (b) 591 bp tcdB gene of Clostridium difficile Lane-1: 100 bp DNA marker; Lane-2: Positive control, Lane-3: Sample 89, Lane-4: Sample 95, Lane-5: Sample 108. Lane-6: Negative control, where sample 95 showed presence of tcdB gene

Click here to view


We reported a higher number of AAD and CDAD cases from MICU; this could be because of the prolonged stay of patients in the intensive care unit and treatment with multiple antibiotics for a longer duration of time. Our study correlates with Lall et al.[19] where a maximum number of cases were from MICU. Shajan et al.[20] also observed maximum IPD cases of CDAD from MICU.

Antibiotics lead to AAD due to disturbance of the composition and function of the normal intestinal flora causing overgrowth of pathogenic microorganisms. In the present study, third-generation cephalosporins followed by fluoroquinolones were the major groups of antibiotics causing both AAD and CDAD. Various studies outside India[21],[22],[23] have quoted the use of cephalosporins as the major group of antibiotics causing C. difficile infection.

Molecular tests are superior to phenotypic methods in the diagnosis of C. difficile infection. In the present study, C. difficile was positive for the species-specific cde gene identified by PCR in 32 (14.41%) of total 222 stool samples. Of these, 18 (56.25%) isolates were toxigenic, i.e., they possessed either the tcdA or the tcdB gene or both. Among the toxigenic isolates, 10 (31.25%) possessed both of the toxigenic genes (tcdA and tcdB) and the remaining 8 (25%) had one of the toxin genes. Only the toxin A (tcdA+ tcdB-) gene was found in 4 (12.5%) and only the toxin B (tcdAv tcdB+) gene in 4 (12.5%) of the toxigenic isolates.

Our study correlates with the study by Vaishnavi et al.[24] where C. difficile was isolated from 174 (15.7%) of a total of 1110 fecal specimens. All the 174 samples were PCR positive; of these, 121 (69.5%) isolates were toxigenic, i.e., they possessed either the tcdA or the tcdB gene or both. Among the toxigenic isolates, 68 (56.2%) possessed both of the toxigenic genes.


  Conclusion Top


Frequent and indiscriminate use of antibiotics has increased the prevalence of CDAD, making it a leading cause of AAD and an important nosocomial infection. Accurate diagnosis of C. difficile infection and detection of toxin-producing strains is essential for optimal treatment and prevention but continues to be challenging.

Financial support and sponsorship

This study was financially supported by KIMSDU for project.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Chaudhry R, Joshy L, Kumar L, Dhawan B. Changing pattern of Clostridium difficile associated diarrhoea in a tertiary care hospital: A 5 year retrospective study. Indian J Med Res 2008;127:377-82.  Back to cited text no. 1
[PUBMED]  [Full text]  
2.
Hedge DD, Strain JD, Heins JR, Farver DK. New advances in the treatment of Clostridium difficile infection (CDI). Ther Clin Risk Manag 2008;4:949-64.  Back to cited text no. 2
    
3.
Vaishnavi C. Established and potential risk factors for Clostridum difficile infection. Indian J Med Microbiol 2009;27:289-300.  Back to cited text no. 3
[PUBMED]  [Full text]  
4.
Kuehne SA, Cartman ST, Heap JT, Kelly ML, Cockayne A, Minton NP. The role of toxin A and toxin B in Clostridium difficile infection. Nature 2010;467:711-3.  Back to cited text no. 4
    
5.
Dhawan B, Chaudhry R. An update on Clostridium difficile infection. Trop Gastroenterol 1997;18:149-52.  Back to cited text no. 5
    
6.
Dhawan B, Chaudhry R, Sharma N. Incidence of Clostridium difficile infection: A prospective study in an Indian hospital. J Hosp Infect 1999;43:275-80.  Back to cited text no. 6
    
7.
Joshy L, Chaudhry R, Dhawan B. Detection and characterization of Clostridium difficile from patients with antibiotic-associated diarrhoea in a tertiary care hospital in North India. J Med Microbiol 2009;58:1657-9.  Back to cited text no. 7
    
8.
Gupta U, Yadav RN. Clostridium difficile in hospital patients. Indian J Med Res 1985;82:398-401.  Back to cited text no. 8
    
9.
Cartman ST, Heap JT, Kuehne SA, Cockayne A, Minton NP. The emergence of 'hypervirulence'in Clostridium difficile. Int J Med Microbiol 2010;300:387-95.  Back to cited text no. 9
    
10.
Goorhuis A, Bakker D, Corver J, Debast SB, Harmanus C, Notermans DW, et al. Emergence of Clostridium difficile infection due to a new hypervirulent strain, polymerase chain reaction ribotype 078. Clin Infect Dis 2008;47:1162-70.  Back to cited text no. 10
    
11.
Collins DA, Hawkey PM, Riley TV. Epidemiology of Clostridium difficile infection in Asia. Antimicrob Resist Infect Control 2013;2:21.  Back to cited text no. 11
    
12.
Warny M, Pepin J, Fang A, Killgore G, Thompson A, Brazier J, et al. Toxin production by an emerging strain of Clostridium difficile associated with outbreaks of severe disease in North America and Europe. Lancet 2005;366:1079-84.  Back to cited text no. 12
    
13.
Vaidhyalingam K, Laxminarayana CS. Internal gas generator system suitable for creating anaerobiosis. Indian J Surg 1980;42:154-9.  Back to cited text no. 13
    
14.
Rinttilä T, Kassinen A, Malinen E, Krogius L, Palva A. Development of an extensive set of 16S rDNA-targeted primers for quantification of pathogenic and indigenous bacteria in faecal samples by real-time PCR. J Appl Microbiol 2004;97:1166-77.  Back to cited text no. 14
    
15.
Spigaglia P, Mastrantonio P. Molecular analysis of the pathogenicity locus and polymorphism in the putative negative regulator of toxin production (TcdC) among Clostridium difficile clinical isolates. J Clin Microbiol 2002;40:3470-5.  Back to cited text no. 15
    
16.
Wren BW, Heard SR, Al-Saleh AI, Tabaqchali S. Characterisation of Clostridium difficile strains by polymerase chain reaction with toxin A- and B-specific primers. J Med Microbiol 1993;38:109-13.  Back to cited text no. 16
    
17.
Vaishnavi C. Clinical spectrum; pathogenesis of Clostridium difficile associated diseases. Indian J Med Res 2010;131:487-99.  Back to cited text no. 17
[PUBMED]  [Full text]  
18.
Segar L, Easow JM, Srirangaraj S, Hanifah M, Joseph NM, Seetha KS. Prevalence of Clostridium difficile; infection among the patients attending a tertiary care teaching hospital. Indian J Pathol Microbiol 2017;60:221-5.  Back to cited text no. 18
[PUBMED]  [Full text]  
19.
Lall S, Nataraj G, Mehta P. Estimation of prevalence and risk factors for Clostridium difficile infection: A neglected pathogen in a tertiary care setting in India. Int J Med Res Rev 2017;5:298-309.  Back to cited text no. 19
    
20.
Shajan SE, Hashim MF, Michael A. Prevalence of Clostridium difficile toxin in diarrhoeal stool samples of patients from a general hospital in eastern province, Saudi Arabia. Int J Med Res Health Sci 2014;3:302-8.  Back to cited text no. 20
    
21.
Dubberke ER, Reske KA, Seiler S, Hink T, Kwon JH, Burnham CA. Risk factors for acquisition and loss of Clostridium difficile colonization in hospitalized patients. Antimicrob Agents Chemother 2015;59:4533-43.  Back to cited text no. 21
    
22.
Slimings C, Riley TV. Antibiotics and hospital-acquired Clostridium difficile infection: Update of systematic review and meta-analysis. J Antimicrob Chemother 2014;69:881-91.  Back to cited text no. 22
    
23.
de Lalla F, Privitera G, Ortisi G, Rizzardini G, Santoro D, Pagano A, et al. Third generation cephalosporins as a risk factor for Clostridium difficile-associated disease: A four-year survey in a general hospital. J Antimicrob Chemother 1989;23:623-31.  Back to cited text no. 23
    
24.
Vaishnavi C, Singh M, Mahmood S, Kochhar R. Prevalence and molecular types of Clostridium difficile isolates from faecal specimens of patients in a tertiary care centre. J Med Microbiol 2015;64:1297-304.  Back to cited text no. 24
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]



 

Top
 
 
  Search
 
Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

 
  In this article
Abstract
Introduction
Materials and Me...
Observation and ...
Discussion
Conclusion
References
Article Figures
Article Tables

 Article Access Statistics
    Viewed38    
    Printed8    
    Emailed0    
    PDF Downloaded14    
    Comments [Add]    

Recommend this journal


[TAG2]
[TAG3]
[TAG4]