Evaluation of detection methods for Arcobacter infections in diarrhea specimens among children under six years in Arak City

Authors
Infectious Diseases Research Center (IDRC), Arak University of Medical Sciences, Arak, IR Iran
Abstract
Background: In recent years, the presence of large amount of research isolating and detecting Arcobacter spp. from animals and humans with diarrhea and from food samples highlights the importance of Arcobacter spp. as emerging food-borne pathogens worldwide. Recently, independent studies have been conducted, making significant progress in the understanding of the classification and pathogenicity of this group of microorganisms. However, the incidence of Arcobacter infection is likely to be underestimated mainly due to the limitations in current detection and identification methods. This study was done to evaluate the effectiveness of staining method (Gram stain using 1% fuchsinein direct smear) versus PCR as the gold standard.
Materials and Methods: A total of 150 stool samples were collected from under 6 years of age children with diarrhea from clinical centers of Arak. Direct smears of samples were stained with a modified gram staining method (1% fuchsin for 5 minutes with heat). Concurrently, PCR amplification method was performed for all DNA samples.
Results: Arcobacter spp. was isolated by PCR from 28 out of 150 stool samples. Direct staining method identified 79 samples as Campylobacter-like organisms with a sensitivity and specificity values of 100 and 65.50%, respectively.
Conclusion: Detection of Campylobacter-like organisms by 1% fuchsin is simple, inexpensive, and fast with high sensitivity and specificity. Laboratories with limited resources can employ modified gram staining method to detect Campylobacteriaceae infection in early stages.

Keywords








































































































































































































1. Vandamme P, Falsen E, Rossau R, Hoste B, Segers P, Tytgat R, et al. Revision of Campylobacter, Helicobacter, and Wolinella taxonomy: emendation of generic descriptions and proposal of Arcobacter gen. nov. International Journal of Systematic and Evolutionary Microbiology. 1991;41(1):88-103.
https://doi.org/10.1099/00207713-41-1-88
 
2. Wesley IV. Arcobacter infections. Handbook of zoonoses, 2nd ed, sec A: bacterial, rickettsial, chlamydial, and mycotic CRC Press, Boca Raton, Fla. 1994:181-90.
 
3. Ho DD, Ault MJ, Ault MA, Murata GH. Campylobacter enteritis: early diagnosis with Gram's stain. Archives of internal medicine. 1982;142(10):1858-60.
https://doi.org/10.1001/archinte.1982.00340230100018
 
4. Jiang Z-D, DuPont HL, Brown EL, Nandy RK, Ramamurthy T, Sinha A, et al. Microbial etiology of travelers' diarrhea in Mexico, Guatemala, and India: importance of enterotoxigenic Bacteroides fragilis and Arcobacter species. Journal of clinical microbiology. 2010;48(4):1417-9.
https://doi.org/10.1128/JCM.01709-09
 
5. Kopilovic B, Ucakar V, Koren N, Krek M, Kraigher A. Waterborne outbreak of acute gastroenteritis in a costal area in Slovenia in June and July 2008. Eurosurveillance. 2008;13(7–9):1-3.
https://doi.org/10.2807/ese.13.34.18957-en
 
6. Lau S, Woo P, Teng J, Leung K, Yuen K. Identification by 16S ribosomal RNA gene sequencing of Arcobacter butzleri bacteraemia in a patient with acute gangrenous appendicitis. Molecular Pathology. 2002;55(3):182.
https://doi.org/10.1136/mp.55.3.182
 
7. Samie A, Obi C, Barrett L, Powell S, Guerrant R. Prevalence of Campylobacter species, Helicobacter pylori and Arcobacter species in stool samples from the Venda region, Limpopo, South Africa: studies using molecular diagnostic methods. Journal of Infection. 2007;54(6):558-66.
https://doi.org/10.1016/j.jinf.2006.10.047
 
8. Woo PC, Chong KT, Leung K-w, Que T-l, Yuen K-y. Identification of Arcobacter cryaerophilus isolated from a traffic accident victim with bacteremia by 16S ribosomal RNA gene sequencing. Diagnostic microbiology and infectious disease. 2001;40(3):125-7.
https://doi.org/10.1016/S0732-8893(01)00261-9
 
9. Rivas L, Fegan N, Vanderlinde P. Isolation and characterisation of Arcobacter butzleri from meat. International journal of food microbiology. 2004;91(1):31-41.
https://doi.org/10.1016/S0168-1605(03)00328-3
 
10. Khoshbakht R, Tabatabaei M, Shirzad Aski H, Seifi S. Occurrence of Arcobacter in Iranian poultry and slaughterhouse samples implicates contamination by processing equipment and procedures. British poultry science. 2014;55(6):732-6.
https://doi.org/10.1080/00071668.2014.971223
 
11. Duffy LL, Fegan N. Prevalence and concentration of Arcobacter spp. on Australian beef carcasses. Journal of food protection. 2012;75(8):1479-82.
https://doi.org/10.4315/0362-028X.JFP-12-093
 
12. Morita Y, Maruyama S, Kabeya H, Boonmar S, Nimsuphan B, Nagai A, et al. Isolation and phylogenetic analysis of Arcobacter spp. in ground chicken meat and environmental water in Japan and Thailand. Microbiology and immunology. 2004;48(7):527-33.
https://doi.org/10.1111/j.1348-0421.2004.tb03548.x
 
13. González A, Ferrús MA. Study of Arcobacter spp. contamination in fresh lettuces detected by different cultural and molecular methods. International journal of food microbiology. 2011;145(1):311-4.
https://doi.org/10.1016/j.ijfoodmicro.2010.11.018
 
14. Akıncıoğlu F. Isolation of arcobacter species from different water sources and characterization of ısolated species by molecular techniques: İzmir Institute of Technology; 2011.
 
15. Diergaardt S, Venter S, Spreeth A, Theron J, Brözel V. The occurrence of campylobacters in water sources in South Africa. Water research. 2004;38(10):2589-95.
https://doi.org/10.1016/j.watres.2004.03.004
 
16. Jacob J, Woodward D, Feuerpfeil I, Johnson W. Isolation of Arcobacter butzleri in raw water and drinking water treatment plants in Germany. Zentralblatt fur Hygiene und Umweltmedizin= International journal of hygiene and environmental medicine. 1998;201(2):189-98.
 
17. Musmanno R, Russi M, Lior H, Figura N. Jv Vjtr () Virulence Factors () F Arco) Ja Cter J3l/Tzj Jjj Strains Iso) Latei) From Superficial, Water Samiples. Microbiologica. 1997;20:63-8.
 
18. Rice E, Rodgers M, Wesley I, Johnson C, Tanner S. Isolation of Arcobacter butzleri from ground water. Letters in Applied Microbiology. 1999;28(1):31-5.
https://doi.org/10.1046/j.1365-2672.1999.00483.x
 
19. Fera M, La Camera E, Carbone M, Malara D, Pennisi M. Pet cats as carriers of Arcobacter spp. in Southern Italy. Journal of applied microbiology. 2009;106(5):1661-6.
https://doi.org/10.1111/j.1365-2672.2008.04133.x
 
20. De Smet S, De Zutter L, Debruyne L, Vangroenweghe F, Vandamme P, Houf K. Arcobacter population dynamics in pigs on farrow-to-finish farms. Applied and environmental microbiology. 2011;77(5):1732-8.
https://doi.org/10.1128/AEM.02409-10
 
21. Houf K, De Smet S, Baré J, Daminet S. Dogs as carriers of the emerging pathogen Arcobacter. Veterinary microbiology. 2008;130(1):208-13.
https://doi.org/10.1016/j.vetmic.2008.01.006
 
22. Vandenberg O, Dediste A, Houf K, Ibekwem S, Souayah H, Cadranel S, et al. Arcobacter species in humans. Emerging infectious diseases. 2004;10(10):1863.
https://doi.org/10.3201/eid1010.040241
 
23. Vandamme P, Vancanneyt M, Pot B, Mels L, Hoste B, Dewettinck D, et al. Polyphasic taxonomic study of the emended genus Arcobacter with Arcobacter butzleri comb. nov. and Arcobacter skirrowii sp. nov., an aerotolerant bacterium isolated from veterinary specimens. International Journal of Systematic and Evolutionary Microbiology. 1992;42(3):344-56.
https://doi.org/10.1099/00207713-42-3-344
 
24. Wilson G, Aitchison L. The use of a combined enrichment–filtration technique for the isolation of Campylobacter spp. from clinical samples. Clinical microbiology and infection. 2007;13(6):643-4.
https://doi.org/10.1111/j.1469-0691.2007.01712.x
 
25. Mshana S, Joloba M, Kakooza A, Kaddu-Mulindwa D. Role of microscopic examination of stool specimens in the diagnosis of campylobacter infection from children with acute diarrhoea in Kampala, Uganda. Tanzania journal of health research. 2010;12(1):100-3.
https://doi.org/10.4314/thrb.v12i1.56367
 
26. Wang H, Murdoch DR. Detection of Campylobacter species in faecal samples by direct Gram stain microscopy. Pathology. 2004;36(4):343-4.
https://doi.org/10.1080/0031302042000224575
 
27. Mushi MF, Paterno L, Tappe D, Deogratius AP, Seni J, Moremi N, et al. Evaluation of detection methods for Campylobacter infections among under-fives in Mwanza City, Tanzania. The Pan African medical journal. 2014;19.
https://doi.org/10.11604/pamj.2014.19.392.4242
 
28. Ghosh R, Uppal B, Aggarwal P, Chakravarti A, Jha AK, Dubey A. A comparative study of conventional and molecular techniques in diagnosis of campylobacter gastroenteritis in children. Annals of Clinical & Laboratory Science. 2014;44(1):42-8.
 
29. Porte L, Varela C, Haecker T, Morales S, Weitzel T. Impact of changing from staining to culture techniques on detection rates of Campylobacter spp. in routine stool samples in Chile. BMC infectious diseases. 2016;16(1):196.
https://doi.org/10.1186/s12879-016-1546-7
 
30. Kayman T, Abay S, Hizlisoy H, Atabay HI, Diker KS, Aydin F. Emerging pathogen Arcobacter spp. in acute gastroenteritis: molecular identification, antibiotic susceptibilities and genotyping of the isolated arcobacters. Journal of medical microbiology. 2012;61(10):1439-44.
https://doi.org/10.1099/jmm.0.044594-0
 
31. Prouzet-Mauléon V, Labadi L, Bouges N, Ménard A, Mégraud F. Arcobacter butzleri: underestimated enteropathogen. Emerging infectious diseases. 2006;12(2):307.
https://doi.org/10.3201/eid1202.050570
 
32. Vandamme P, Pugina P, Benzi G, Van Etterijck R, Vlaes L, Kersters K, et al. Outbreak of recurrent abdominal cramps associated with Arcobacter butzleri in an Italian school. Journal of Clinical Microbiology. 1992;30(9):2335-7.
 
33. Taylor DN, Perlman DM, Echeverria PD, Lexomboon U, Blaser MJ. Campylobacter immunity and quantitative excretion rates in Thai children. Journal of Infectious Diseases. 1993;168(3):754-8.
https://doi.org/10.1093/infdis/168.3.754