Enterobius Vermicularis Ova Descriptive Essay

This article is about the human infection with pinworms. For the organism itself, see Pinworm (parasite).

Pinworm infection, also known as enterobiasis, is a human parasitic disease caused by the pinworm.[3] The most common symptom is itching in the anal area.[1] This can make sleeping difficult.[1] The period of time from swallowing eggs to the appearance of new eggs around the anus is 4 to 8 weeks.[2] Some people who are infected do not have symptoms.[1]

The disease is spread between people by pinworm eggs.[1] The eggs initially occur around the anus and can survive for up to three weeks in the environment.[1] They may be swallowed following contamination of the hands, food, or other articles.[1] Those at risk are those who go to school, live in a health care institution or prison, or take care of people who are infected.[1] Other animals do not spread the disease.[1] Diagnosis is by seeing the worms which are about one centimeter or the eggs under a microscope.[1][6]

Treatment is typically with two doses of the medications mebendazole, pyrantel pamoate, or albendazole two weeks apart.[4] Everyone who lives with or takes care of an infected person should be treated at the same time.[1] Washing personal items in hot water after each dose of medication is recommended.[1] Good handwashing, daily bathing in the morning, and daily changing of underwear can help prevent reinfection.[1]

Pinworm infections commonly occur in all parts of the world.[1][5] It is the most common worm infection in the developed world.[1] School aged children are the most commonly infected.[1] In the United States about 20% of people at one point in time develop pinworm.[3] Infection rates among high risk groups may be as high as 50%.[2] It is not considered a serious disease.[5] Pinworms are believed to have affected humans throughout history.[7]

Signs and symptoms[edit]

One third of individuals with pinworm infection are totally asymptomatic.[8] The main symptoms are pruritus ani and perinealpruritus, i.e., itching in and around the anus and around the perineum.[8][9][10] The itching occurs mainly during the night,[9][11] and is caused by the female pinworms migrating to lay eggs around the anus.[12][10] Both the migrating females and the clumps of eggs are irritating, but the mechanisms causing the intense pruritus have not been explained.[11] The intensity of the itching varies, and it can be described as tickling, crawling sensations, or even acute pain.[13] The itching leads to continuously scratching the area around the anus, which can further result in tearing of the skin and complications such as secondary bacterial infections, including bacterialdermatitis (i.e., skin inflammation) and folliculitis (i.e., hair follicle inflammation).[9][10][13] General symptoms are insomnia (i.e., persistent difficulties to sleep) and restlessness.[9] A considerable proportion of children suffer from loss of appetite, weight loss, irritability, emotional instability, and enuresis (i.e., inability to control urination).[9]

Pinworms cannot damage the skin,[14] and they do not normally migrate through tissues.[10] However, in women they may move onto the vulva and into the vagina, from there moving to the external orifice of the uterus, and onwards to the uterine cavity, fallopian tubes, ovaries, and peritoneal cavity.[14] This can cause vulvovaginitis, i.e. an inflammation of the vulva and vagina.[9][10] This causes vaginal discharge and pruritus vulvae, i.e., itchiness of the vulva.[9] The pinworms can also enter the urethra, and presumably, they carry intestinal bacteria with them.[14] According to Gutierrez (2000), a statistically significant correlation between pinworm infection and urinary tract infections has been shown;[14] however, Burkhart & Burkhart (2005) maintain that the incidence of pinworms as a cause of urinary tract infections remains unknown.[8] Incidentally, one report indicated that 36% of young girls with a urinary tract infection also had pinworms.[8]Dysuria (i.e., painful urination) has been associated with pinworm infection.[8]

The relationship between pinworm infestation and appendicitis has been researched, but there is a lack of clear consensus on the matter: while Gutierres (2005) maintains that there exists a consensus that pinworms do not produce the inflammatory reaction,[15] Cook (1994) states that it is controversial whether pinworms are causatively related to acute appendicitis,[13] and Burkhart & Burkhart (2004) state that pinworm infection causes symptoms of appendicitis to surface.[8]

Cause[edit]

The cause of a pinworm infection is the worm Enterobius vermicularis. The entire lifecycle — from egg to adult — takes place in the human gastrointestinal tract of a single human host.[12][16] Cook et al. (2009) and Burkhart & Burkhart (2005) disagree over the length of this process, with Cook et al. stating two to four weeks,[9] while Burkhart & Burkhart states that it takes from four to eight weeks.[17]

Spread[edit]

Pinworm infection spreads through human-to-human transmission, by ingesting (i.e., swallowing) infectious pinworm eggs.[17][18] The eggs are hardy and can remain viable (i.e., infectious) in a moist environment for up to three weeks,[11][17] though in a warm dry environment they usually last only 1–2 days.[19] They do not tolerate heat well, but can survive in low temperatures: at −8 degrees Celsius (18 °F), two-thirds of the eggs are still viable after 18 hours.[11]

After the eggs have been initially deposited near the anus, they are readily transmitted to other surfaces through contamination.[18] The surface of the eggs is sticky when laid,[12][11] and the eggs are readily transmitted from their initial deposit near the anus to fingernails, hands, night-clothing and bed linen.[9] From here, eggs are further transmitted to food, water, furniture, toys, bathroom fixtures and other objects.[12][17][18] Household pets often carry the eggs in their fur, while not actually being infected.[20] Dust containing eggs can become airborne and widely dispersed when dislodged from surfaces, for instance when shaking out bed clothes and linen.[11][17][20] Consequently, the eggs can enter the mouth and nose through inhalation, and be swallowed later.[9][11][17][18] Although pinworms do not strictly multiply inside the body of their human host,[9] some of the pinworm larvae may hatch on the anal mucosa, and migrate up the bowel and back into the gastrointestinal tract of the original host.[9][17] This process is called retroinfection.[11][17] According to Burkhart (2005), when this retroinfection occurs, it leads to a heavy parasitic load and ensures that the pinworm infestation continues.[17] This statement is contradictory to a statement by Caldwell, who contends that retroinfection is rare and not clinically significant.[11] Despite the limited, 13-week lifespan of individual pinworms,[12]autoinfection (i.e., infection from the original host to itself), either through the anus-to-mouth route or through retroinfection, causes the pinworms to inhabit the same host indefinitely.[17]

Life cycle[edit]

The life cycle begins with eggs being ingested.[12] The eggs hatch in the duodenum (i.e., first part of the small intestine).[18] The emerging pinworm larvae grow rapidly to a size of 140 to 150 micrometers in size,[9] and migrate through the small intestine towards the colon.[12] During this migration they moult twice and become adults.[12][17] Females survive for 5 to 13 weeks, and males about 7 weeks.[12] The male and female pinworms mate in the ileum (i.e., last part of the small intestine),[12] whereafter the male pinworms usually die,[18] and are passed out with stool.[11] The gravid female pinworms settle in the ileum, caecum (i.e., beginning of the large intestine), appendix and ascending colon,[12] where they attach themselves to the mucosa[17] and ingest colonic contents.[10] Almost the entire body of a gravid female becomes filled with eggs.[18] The estimations of the number of eggs in a gravid female pinworm ranges from about 11,000[12] to 16,000.[17] The egg-laying process begins approximately five weeks after initial ingestion of pinworm eggs by the human host.[12] The gravid female pinworms migrate through the colon towards the rectum at a rate of 12 to 14 centimeters per hour.[12] They emerge from the anus, and while moving on the skin near the anus, the female pinworms deposit eggs either through (1) contracting and expelling the eggs, (2) dying and then disintegrating, or (3) bodily rupture due to the host scratching the worm.[18] After depositing the eggs, the female becomes opaque and dies.[11] The reason the female emerges from the anus is to obtain the oxygen necessary for the maturation of the eggs.[11]

Diagnosis[edit]

Diagnosis depends on finding the eggs or the adult pinworms.[18] Individual eggs are invisible to the naked eye, but they can be seen using a low-power microscope.[20] On the other hand, the light-yellowish thread-like adult pinworms are clearly visually detectable, usually during the night when they move near the anus, or on toilet paper.[8][13][20] Transparent adhesive tape (e.g. Scotch Tape) applied on the anal area will pick up deposited eggs, and diagnosis can be made by examining the tape with a microscope.[15][20] This test is most successful if done every morning for several days, because the females do not lay eggs every day, and the number of eggs vary.[20]

Pinworms do not lay eggs in the feces,[20] but sometimes eggs are deposited in the intestine.[18] As such, routine examination of fecal material gives a positive diagnosis in only 5 to 15% of infected subjects,[13] and is therefore of little practical diagnostic use.[9] In a heavy infection, female pinworms may adhere to stools that pass out through the anus, and they may thus be detected on the surface on the stool.[13][18] Adult pinworms are occasionally seen during colonoscopy.[13] On a microscopic level, pinworms have an identifying feature of alae (i.e., protruding ridges) running the length of the worm.[21]

Prevention[edit]

Pinworm infection cannot be totally prevented under most circumstances.[22] This is due to the prevalence of the parasite and the ease of transmission through soiled night clothes, airborne eggs, contaminated furniture, toys and other objects.[18] Infection may occur in the highest strata of society, where hygiene and nutritional status are typically high.[23] The stigma associated with pinworm infection is hence considered a possible over-emphasis.[23]Counselling is sometimes needed for upset parents that have discovered their children are infected, as they may not realize how prevalent the infection is.[18]

Preventative action revolves around personal hygiene and the cleanliness of the living quarters.[23] The rate of reinfection can be reduced through hygienic measures, and this is recommended especially in recurring cases.[20][23] The main measures are keeping fingernails short, and washing and scrubbing hands and fingers carefully, especially after defecation and before meals.[23][24] Under ideal conditions, bed covers, sleeping garments, and hand towels should be changed daily.[23] Simple laundering of clothes and linen disinfects them.[23] Children should wear gloves while asleep, and the bedroom floor should be kept clean.[23] Food should be covered to limit contamination with dust-borne parasite eggs.[23] Household detergents have little effect on the viability of pinworm eggs, and cleaning the bathroom with a damp cloth moistened with an antibacterial agent or bleach will merely spread the still-viable eggs.[23] Similarly, shaking clothes and bed linen will detach and spread the eggs.[23]

Treatment[edit]

Medication is the primary treatment for pinworm infection.[23] They are so effective that many medical scientists regard hygienic measures as impractical.[20] However, reinfection is frequent regardless of the medication used.[8] Total elimination of the parasite in a household may require repeated doses of medication for up to a year or more.[9] Because the drugs kill the adult pinworms, but not the eggs, the first retreatment is recommended in two weeks.[20] Also, if one household member spreads the eggs to another, it will be a matter of two or three weeks before those eggs become adult worms and thus amenable to treatment.[24]Asymptomatic infections, often in small children, can serve as reservoirs of infection, and therefore the entire household should be treated regardless of whether or not symptoms are present.[9][23]

The benzimidazole compounds albendazole (brand names e.g., Albenza, Eskazole, Zentel and Andazol) and mebendazole (brand names e.g., Ovex, Vermox, Antiox and Pripsen) are the most effective.[23] They work by inhibiting the microtubule function in the pinworm adults, causing glycogen depletion,[23] thereby effectively starving the parasite.[24] A single 100 milligram dose of mebendazole with one repetition after a week, is considered the safest, and is usually effective with cure rate of 96%.[8][23] Mebendazole has no serious side effects, although abdominal pain and diarrhea have been reported.[23]Pyrantel pamoate (also called pyrantel embonate, brand names e.g., Reese's Pinworm Medicine, Pin-X, Combantrin, Anthel, Helmintox, and Helmex) kills adult pinworms through neuromuscular blockade,[24] and is considered as effective as the benzimidazole compounds and is used as a second-line medication.[9] Other medications are piperazine, which causes flaccid paralysis in the adult pinworms, and pyrvinium pamoate (also called pyrvinium embonate), which works by inhibiting oxygen uptake of the adult pinworms.[24] Pinworms located in the genitourinary system (in this case, female genital area) may require other drug treatments.[8]

Epidemiology[edit]

Pinworm infection occurs worldwide,[10] and is the most common helminth (i.e., parasitic worm) infection in the United States and Western Europe.[17] In the United States, a study by the Center of Disease Control reported an overall incidence rate of 11.4% among people of all ages.[17] Pinworms are particularly common in children, with prevalence rates in this age group having been reported as high as 61% in India, 50% in England, 39% in Thailand, 37% in Sweden, and 29% in Denmark.[17]Finger sucking has been shown to increase both incidence and relapse rates,[17] and nail biting has been similarly associated.[13] Because it spreads from host to host through contamination, enterobiasis is common among people living in close contact, and tends to occur in all people within a household.[10] The prevalence of pinworms is not associated with gender,[10] nor with any particular social class, race, or culture.[17] Pinworms are an exception to the tenet that intestinal parasites are uncommon in affluent communities.[17]

History[edit]

The earliest known instance of pinworms is evidenced by pinworm eggs found in coprolite, carbon dated to 7837 BC at western Utah.[12] Pinworm infection is not classified as a neglected tropical disease unlike many other parasitic worm infections.[25]

Garlic has been used as a treatment in the ancient cultures of China, India, Egypt, and Greece.[26]Hippocrates (459–370 BC) mentioned garlic as a remedy against intestinal parasites.[27] German botanist Lonicerus (1564) recommended garlic against parasitic worms.[28] Applying raw garlic on skin may cause a chemical burn.[29][30]

Notes[edit]

References
  • Hasegawa H, Ikeda Y, Fujisaki A, et al. (December 2005). "Morphology of chimpanzee pinworms, Enterobius (Enterobius) anthropopitheci (Gedoelst, 1916) (Nematoda: Oxyuridae), collected from chimpanzees, Pan troglodytes, on Rubondo Island, Tanzania". The Journal of Parasitology. 91 (6): 1314–7. doi:10.1645/GE-569R.1. PMID 16539010. 
  • "Pinworm". Encyclopædia Britannica. Retrieved 2009-04-08. 
  • "Enterobiasis". Merriam-Webster's Medical Dictionary. Merriam-Webster. Retrieved 2009-04-08. 
  • "Oxyuriasis". Merriam-Webster's Medical Dictionary. Merriam-Webster. Retrieved 2009-04-08. 
  • Totkova A, Klobusicky M, Holkova R, Valent M (2003). "Enterobius gregorii—reality or fiction?"(PDF). Bratislavské Lekárske Listy. 104 (3): 130–133. PMID 12940699. 
  • "Enterobius". NCBI taxonomy database. National Center for Biotechnology Information, U.S. National Library of Medicine. 2009. Retrieved 2009-04-08. 
  • "Enterobiasis". DPDx. Division of Parasitic Diseases, Centers for Disease Control and Prevention. Retrieved 2009-04-08. 
  • Nakano T, Okamoto M, Ikeda Y, Hasegawa H (December 2006). "Mitochondrial cytochrome c oxidase subunit 1 gene and nuclear rDNA regions of Enterobius vermicularis parasitic in captive chimpanzees with special reference to its relationship with pinworms in humans". Parasitology Research. 100 (1): 51–7. doi:10.1007/s00436-006-0238-4. PMID 16788831. 
  • Hugot JP (1983). "Enterobius gregorii (Oxyuridae, Nematoda), a new human parasite". Annales de Parasitologie Humaine et Comparée (in French). 58 (4): 403–4. doi:10.1051/parasite/1983584403. PMID 6416131. 
  • Hasegawa H, Takao Y, Nakao M, Fukuma T, Tsuruta O, Ide K (February 1998). "Is Enterobius gregorii Hugot, 1983 (Nematoda: Oxyuridae) a distinct species?". Journal of Parasitology. 84 (1): 131–4. doi:10.2307/3284542. JSTOR 3284542. PMID 9488350. 
  • Gutiérrez, Yezid (2000). Diagnostic pathology of parasitic infections with clinical correlations(PDF) (Second ed.). Oxford University Press. pp. 354–366. ISBN 0-19-512143-0. Retrieved 21 August 2009. 
  • Cook, Gordon C; Zumla, Alimuddin I. (2009). Manson's tropical diseases (Twentysecond ed.). Saunders Elsevier. pp. 1515–1519. ISBN 978-1-4160-4470-3. Retrieved 18 November 2009. 
  • "B80: Enterobiasis". International Statistical Classification of Diseases and Related Health Problems (ICD) 10th Revision. World Health Organization. 2007. Retrieved 2009-12-05. 
  • Cook GC (September 1994). "Enterobius vermicularis infection". Gut. 35 (9): 1159–62. doi:10.1136/gut.35.9.1159. PMC 1375686. PMID 7959218.
Two female pinworms next to a ruler. The markings are one millimeter apart.
  1. ^ abcdefghijklmnopqrstu"Pinworm Infection FAQs". CDC. 10 January 2013. Archived from the original on 15 October 2016. Retrieved 16 October 2016. 
  2. ^ abc"Epidemiology & Risk Factors". CDC. 10 January 2013. Archived from the original on 18 October 2016. Retrieved 16 October 2016. 
  3. ^ abcStermer, E; Sukhotnic, I; Shaoul, R (May 2009). "Pruritus ani: an approach to an itching condition". Journal of Pediatric Gastroenterology and Nutrition. 48 (5): 513–6. doi:10.1097/mpg.0b013e31818080c0. PMID 19412003. 
  4. ^ ab"Treatment". CDC. 23 September 2016. Archived from the original on 18 October 2016. Retrieved 16 October 2016. 
  5. ^ abcdGriffiths, Christopher; Barker, Jonathan; Bleiker, Tanya; Chalmers, Robert; Creamer, Daniel (2016). Rook's Textbook of Dermatology, 4 Volume Set (9 ed.). John Wiley & Sons. p. 33.13. ISBN 9781118441176. Archived from the original on 5 November 2017. 
  6. ^"Biology". CDC. 10 January 2013. Archived from the original on 18 October 2016. Retrieved 16 October 2016. 
  7. ^Bynum, W. F.; Porter, Roy (2013). Companion Encyclopedia of the History of Medicine. Routledge. p. 358. ISBN 9781136110368. Archived from the original on 5 November 2017. 
  8. ^ abcdefghijBurkhart & burkhart 2005, p. 838
  9. ^ abcdefghijklmnopqCook et al. 2009, p. 1516
  10. ^ abcdefghiGutiérrez 2005, p. 355.
  11. ^ abcdefghijklCaldwell 1982, p. 307.
  12. ^ abcdefghijklmnoCook 1994, p. 1159
  13. ^ abcdefghCook 1994, p. 1160
  14. ^ abcdGutiérrez 2005, p. 356.
  15. ^ abGutiérrez 2005, p. 363.
  16. ^Gutiérrez 2005, p. 354.
  17. ^ abcdefghijklmnopqrsBurkhart & burkhart 2005, p. 837
  18. ^ abcdefghijklmGarcia 1999, p. 246
  19. ^Cook, G C (1994). "Enterobius vermicularis infection". Gut. 35 (9): 1159–1162. doi:10.1136/gut.35.9.1159. ISSN 0017-5749. PMC 1375686. PMID 7959218. 
  20. ^ abcdefghijCaldwell 1982, p. 308.
  21. ^dpdx 2009
  22. ^Garcia 1999, p. 247
  23. ^ abcdefghijklmnopqCook 1994, p. 1161
  24. ^ abcdeCaldwell 1982, p. 309.
  25. ^"Fact sheets: neglected tropical diseases". World Health Organization. WHO Media Centre. Archived from the original on 7 December 2014. Retrieved 6 December 2014. 
  26. ^Petrovska BB, Cekovska S (2010). "Extracts from the history and medical properties of garlic". Pharmacognosy Reviews. 4 (7): 106–10. doi:10.4103/0973-7847.65321. PMC 3249897. PMID 22228949. 
  27. ^Tucakov J. Beograd: Naucna knjiga; 1948. Farmakognozija; pp. 278–80.
  28. ^3. Tucakov J. Beograd: Kultura; 1971. Lecenje biljem - fitoterapija; pp. 180–90.
  29. ^Borrelli, F; Capasso, R; Izzo, AA (November 2007). "Garlic (Allium sativum L.): adverse effects and drug interactions in humans". Molecular Nutrition & Food Research. 51 (11): 1386–97. doi:10.1002/mnfr.200700072. PMID 17918162. 
  30. ^Friedman, T; Shalom, A; Westreich, M (October 2006). "Self-inflicted garlic burns: our experience and literature review". International Journal of Dermatology. 45 (10): 1161–3. doi:10.1111/j.1365-4632.2006.02860.x. PMID 17040429. 

Enterobius vermicularis: ancient DNA from north and south American human coprolites

 

 

Alena M IñiguezI; Karl J ReinhardII; Adauto AraújoIII; Luiz Fernando FerreiraIII; Ana Carolina P VicenteI

I Laboratório de Genética Molecular de Microorganismos, Departamento de Genética, Instituto Oswaldo Cruz, Fiocruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brasil
IISchool of Natural Resource Sciences, University of Nebraska, Lincoln, NE, USA
IIIEscola Nacional de Saúde Pública, Fiocruz, Rio de Janeiro, RJ, Brasil

Address to correspondence

 

 


ABSTRACT

A molecular paleoparasitological diagnostic approach was developed for Enterobius vermicularis. Ancient DNA was extracted from 27 coprolites from archaeological sites in Chile and USA. Enzymatic amplification of human mtDNA sequences confirmed the human origin. We designed primers specific to the E. vermicularis 5S ribosomal RNA spacer region and they allowed reproducible polymerase chain reaction identification of ancient material. We suggested that the paleoparasitological microscopic identification could accompany molecular diagnosis, which also opens the possibility of sequence analysis to understand parasite-host evolution.

Key words: ancient DNA - Enterobius vermicularis - coprolites


 

 

Based on several lines of evidence including archaeoparasitology and cladistic analysis, the pinworm Enterobius vermicularis is one of the most ancient parasites of humans and has a pre-hominid evolutionary origin (Ferreira et al. 1997, Hugot et al. 1999). Paleoparasito-logical studies showed the presence of pinworm eggs in 10,000-year-old human coprolites from United States and in coprolites from Chile and Peru dating from 2200 to 400 BC (Ferreira et al. 1997). In some prehistoric cultures, E. vermicularis reached very high prevalence as indicated by the numbers of coprolites that contain eggs (Reinhard 1998). Until now, diagnosis of pinworms in archaeological remains was dependent on microscopic examination. This is a particularly poor method of identifying prehistoric pinworm infections (Reinhard 1990, Araújo et al. 1998). Recent work revealing the ancient DNA (aDNA) of parasites such as Trypanosoma cruzi in mummies and Ascaris in coprolites (Ferreira et al. 2000, Loreille et al. 2001) opened this area of investigation to other organisms. This paper presents a molecular diagnosis of E. vermicularis using the conserved region from 5S ribosomal RNA (rRNA) intergenic spacer as target.

The coprolites (n = 27) from archaeological sites in Chile and North America were analyzed. Samples from Chile were collected in archaeological sites of Caserones (n = 2), Tarapacá Valley, dating from 400 BC to 800 AD (Ferreira et al. 1984); of Tulan (n = 20), San Pedro de Atacama dating to 1000 BC (Ferreira et al. 1989); and of Tiliviche (n = 2) dated from 4110 to 1950 BC (Araújo et al. 1983). USA samples (n = 3) were from Antelope House, an Anasazi village site in Canyon de Chelly, Arizona dating from 900 AD (Reinhard 1996) (Table). DNA extraction and amplification were performed under the procedures established for working with aDNA to avoid contamination with modern molecules (Hofreiter et al. 2001, Marota & Rollo 2002). The surface of the samples was exposed to UV light and the coprolite core was ground. Coprolite powder (5-2 g) was hydrated in ddH2O or TE buffer (Tris-HCl 10mM, EDTA 1mM, pH 8.0). Coprolites, formerly used in microscopic diagnostic in 0.5% trisodium phosphate aqueous solution (Ferreira et al. 1989), also were analyzed. Sediments of 150 µl were treated by 72 h with 400 µl digestion buffer (NaCl 100 mM, Tris-HCl 50 mM, SDS 1%, EDTA 50 mM, pH 8.0), and added 20 µl DTT 1M, 60 µl proteinase K 10 mg/ml (Gibco BRL) and 100 µl SDS10%. The reactions were incubated at 55-60oC for 3-24 h with occasional homogenization followed by the phenol/chloroform extraction and purified using silica resin column (Glass Max DNA Isolation Spring Cartridge System Gibco-BRL).

In order to determine the nature of the coprolites, human mitochondrial DNA (mtDNA) amplification was done using the procedure described by Pääbo (1990) and Handt et al. (1996). E. vermicularis amplification was performed by nested PCR targeting E. vermicularis 5S rRNA spacer region: Entf (5'-CACTTGCTATACCAACAACAC-3') and Entr (5'-GCGCTACTAAACCATAGAG-3'); and internal Eva (5'-ACAACACTTGCACGTCTC-3') Evb (5'-GAATTGCTCGTTTGC-3'). PCR final volume reaction was: 25 µl using 20 mM Tris-HCl, 0.5 µM KCl (Gibco BRL 10X Buffer), 2 mM MgCl2, 0.2 mM each dNTPs, 1mg/ml bovine serum albumin (BSA) and 500 ng of each oligonucleotide. The mixture was exposed to 30 min UV radiation before the 2.5 U of Taq polymerase (Gibco BRL) and 50-100 ng DNA extract addition. The reactions were subjected to an initial cycle of 5 min at 94oC, followed by 35 cycles of 94oC for 1min, 50-55oC for 30 sec and 72oC for 30 sec in a programmable thermal controller (PTC100 60 v, MJ Research, Inc). Extraction and negative PCR controls were included. Amplicons were hybridized with a radiolabeled probe from the same region amplified from a modern sample (Sambrook et al. 1989).

Human mtDNA was retrieved from almost all coprolite samples by using Handt et al. (1996) procedure (Fig. 1). Due to the nature of aDNA, the target choice is a crucial step for the successful sequence retrieval. The complete pinworm highly conserved ribossomal 5S intergenic region is about 800 bp (Liu et al. 1995), with several copies in an organism. We designed primers for a nested PCR targeting this region. The first and second primer pair produced 420 bp and 198 bp specific and unique amplicon, respectively. The control of primer specificity was done previously using DNA extracted from modern feces and E. vermicularis experimental coprolites (Iñiguez 1998). We were successful in the molecular paleoparasitological diagnosis of E. vermicularis using the specific pinworm 5S rRNA spacer region (Fig. 2). The hybridization result confirmed the specific nature of the diagnostic bands with 198 bp length (data not shown). All positive samples in the microscopic analysis but nine were PCR positive. Two samples, 706 and 716, negative in the microscopic analysis, yielded E. vermicularis diagnostic amplicon (Table). The sample 721 was PCR negative either to mtDNA or pinworm target. Considering that during this work the experimental procedure of DNA extraction, purification and the set up of PCR reactions, were done in two different laboratories and repeated at least twice, we concluded that the aDNA 721 was highly degraded (Lindahl 1993, Marota et al. 2002). In this regard, microscopic identification of pinworms eggs is still relevant for paleoparasitolo-gical diagnosis. However, molecular approach not only offers a precise identification, but also the opportunity of ancient parasite sequence comparisons with those of contemporary populations. Further analysis of human mtDNA and pinworm aDNAs sequences can provide more comprehension about E. vermicularis evolution and their human host.

 

Fig. 1: mitochondrial human DNA amplicom (185 bp): Lanes - 1 to 12: coprolite samples: 168, 170, 384, 385, 704, 706, 708, 714, 715, 716, 719, and 721, respectively. Lane 13: PCR negative control; 14: 100 bp DNA ladder (Gibco BRL). (2 % Agarose gel electrophoresis).

 

 

Fig. 2: Enterobius vermicularis diagnostic amplicon (198 bp). Lanes - 1 to 12: coprolite samples 170, 385, 704, 708, 714, 721, 168, 384, 706, 715, 716 and 719, respectively; 13: PCR negative control; 14: 1 kb DNA ladder (Gibco BRL). (2 % Agarose gel electrophoresis).

 

 

 

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Address to correspondence
Alena M Iñiguez
Fax: +55-21-2260.4282.
E-mail: alena@ioc.fiocruz.br

Received 26 August 2002
Accepted 25 November 2002
This study was supported by ENSP-Fiocruz, IOC-Fiocruz and Fulbright Comission.

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