• 1.

    Liu L, Johnson HL, Cousens S, Perin J, Scott S, Lawn JE, Rudan I, Campbell H, Cibulskis R, Li M, Mathers C, Black RE; Child Health Epidemiology Reference Group of WHO and UNICEF, 2012. Global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since 2000. Lancet 379: 21512161.

    • Search Google Scholar
    • Export Citation
  • 2.

    UNICEF, WHO, 2009. Diarrhoea: Why Children are Still Dying and What Can Be Done. New York, NY: United Nations Children's Fund. Available at: http://www.who.int/child_adolescent_health/documents/who. Accessed October 6, 2014.

    • Search Google Scholar
    • Export Citation
  • 3.

    Das SK, Faruque AS, Chisti MJ, Malek MA, Salam MA, Sack DA, 2012. Changing trend of persistent diarrhoea in young children over two decades: observations from a large diarrheal disease hospital in Bangladesh. Acta Paediatr 101: e452e457.

    • Search Google Scholar
    • Export Citation
  • 4.

    World Health Organization and United Nations Children's Fund, 2013. Ending Preventable Child Deaths from Pneumonia and Diarrhea by 2025. The Integrated Global Action Plan for Pneumonia and Diarrhea (GAPPD). Geneva, Switzerland and New York, NY: World Health Organization and United Nations Children's Fund. Available at: http://apps.who.int/iris/bitstream/10665/79200/1/9789241505239_eng.pdf?ua=1. Accessed October 4, 2014.

    • Search Google Scholar
    • Export Citation
  • 5.

    Kotloff KL, Nataro JP, Blackwelder WC, Nasrin D, Farag TH, Panchalingam S, Wu Y, Sow SO, Sur D, Breiman RF, Faruque ASG, Zaidi AKM, Saha D, Alonso PL, Tamboura B, Sanogo D, Onwuchekwa U, Manna B, Ramamurthy T, Kanungo S, Ochieng JB, Omore R, Oundo JO, Hossain A, Das SK, Ahmed S, Qureshi S, Quadri F, Adegbola RA, Antonio M, Hossain MJ, Akinsola A, Mandomando I, Nhampossa T, Acácio S, Biswas K, O'Reilly CE, Mintz ED, Berkeley LY, Muhsen K, Sommerfelt H, Robins-Browne RM, Levine MM, 2013. Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study. Lancet 382: 209222.

    • Search Google Scholar
    • Export Citation
  • 6.

    Rahman AE, Moinuddin M, Molla M, Worku A, Hurt L, Kirkwood B, Mohan SB, Mazumder S, Bhutta Z, Raza F, Mrema S, Masanja H, Kadobera D, Waiswa P, Bahl R, Zangenbergh M, Muheh L, 2014. Childhood diarrheal deaths in seven low- and middle-income countries. Bull World Health Organ 92: 664671.

    • Search Google Scholar
    • Export Citation
  • 7.

    Samie A, El Bakri A, Ra'ed Abu Odeh R, 2012. Amoebiasis in the Tropics: Epidemiology and Pathogenesis. Available at: www.intechopen.com. Accessed October 10, 2014.

    • Search Google Scholar
    • Export Citation
  • 8.

    Guerrant RL, Gilder TV, Steiner TS, Thielman NM, Slutsker L, Tauxe RV, Hennessy T, Griffin PM, DuPont H, Sack RB, Tarr P, Neill M, Nachamkin I, Reller LB, Osterholm MT, Bennish ML, Pickering LK, 2001. Practice guidelines for the management of infectious diarrhea. CID 32: 231251.

    • Search Google Scholar
    • Export Citation
  • 9.

    Morris AJ, Murray PR, Reller LB, 1996. Contemporary testing for enteric pathogens: the potential for cost, time, and health care savings. J Clin Microbiol 34: 17761778.

    • Search Google Scholar
    • Export Citation
  • 10.

    Hines J, Nachamkin I, 1996. Effective use of the clinical microbiology laboratory for diagnosing diarrheal diseases. Clin Infect Dis 23: 12921301.

    • Search Google Scholar
    • Export Citation
  • 11.

    González-Ruiz A, Haque R, Aguirre A, Castañón G, Hall A, Guhl F, Ruiz-Palacios G, Miles MA, Warhurst DC, 1994. Value of microscopy in the diagnosis of dysentery associated with invasive Entamoeba histolytica. J Clin Pathol 47: 236239.

    • Search Google Scholar
    • Export Citation
  • 12.

    Tanyuksel M, Petri WA Jr, 2003. Laboratory diagnosis of amebiasis. Clin Microbiol Rev 16: 713729.

  • 13.

    Stark FRD, Beebe N, Marriott D, Elli J, Harkness J, 2007. A review of laboratory diagnostic techniques for Entamoeba species. Clin Microbiol Rev 20: 511532.

    • Search Google Scholar
    • Export Citation
  • 14.

    Haque R, Ali IK, Akther S, Petri WA Jr, 1998. Comparison of PCR, isoenzyme analysis, and antigen detection for diagnosis of E. histolytica infection. J Clin Microbiol 36: 449452.

    • Search Google Scholar
    • Export Citation
  • 15.

    Mirelman D, Nuchamowitz Y, Stolarsky T, 1997. Comparison of use of enzyme-linked immunosorbent assay-based kits and PCR amplification of rRNA genes for simultaneous detection of E. histolytica and E. dispar. J Clin Microbiol 35: 24052407.

    • Search Google Scholar
    • Export Citation
  • 16.

    Roy S, Kabir M, Mondal D, Ali IK, Petri WA Jr, Haque R, 2005. Real-time-PCR assay for diagnosis of E. histolytica infection. J Clin Microbiol 43: 21682172.

    • Search Google Scholar
    • Export Citation
  • 17.

    World Health Organization, 1997. Amebiasis. WHO Wkly Epidemiol Rec 72: 97100.

  • 18.

    Okeke IN, Ojo O, Lamikanra A, Kaper JB, 2003. Etiology of acute diarrhea in adults in southwestern Nigeria. J Clin Microbiol 41: 45254530.

  • 19.

    Idowu OA, Rowland SA, 2006. Oral fecal parasites and personal hygiene of food handlers in Abeokuta, Nigeria. Afr Health Sci 6: 160164.

  • 20.

    Oyerinde JPO, Alonge AA, Adegbite-Hollist AF, Ogunbi O, 1979. The epidemiology of Entamoeba histolytica in a Nigerian urban population. Int J Epidemiol 8: 5560.

    • Search Google Scholar
    • Export Citation
  • 21.

    Ilero Town in Nigeria. Available at: http://www.fallingrain.com/world/NI/00/Ilero.html. Accessed May 20, 2015.

  • 22.

    Smith B, Li N, Andersen AS, Slotved HC, Krogfelt KA, 2011. Optimising bacterial DNA extraction from faecal samples: comparison of three methods. Open Microbiol J 5: 1417.

    • Search Google Scholar
    • Export Citation
  • 23.

    Verweij JJ, Oostvogel F, Brienen EAT, Nang-Beifubah A, Ziem J, Polderman AM, 2003. Prevalence of E. histolytica and E. dispar in northern Ghana. Trop Med Int Health 8: 11531156.

    • Search Google Scholar
    • Export Citation
  • 24.

    Verweij JJ, Blangé RA, Templeton K, Schinkel J, Brienen EAT, van Rooyen MAA, van Lieshout L, Poldermanet AM, 2004. Simultaneous detection of E. histolytica, Giardia lamblia, and Cryptosporidium parvum in fecal samples by using multiplex real-time PCR. J Clin Microbiol 42: 12201223.

    • Search Google Scholar
    • Export Citation
  • 25.

    McElligott JT, Naaktgeboren C, Makuma-Massa H, Summer AP, Deal JL, 2013. Prevalence of intestinal protozoa in communities along the Lake Victoria region of Uganda. Int J Infect Dis 17: 658659.

    • Search Google Scholar
    • Export Citation
  • 26.

    Kebede A, Verweij JJ, Endeshaw T, Messele T, Tasew G, Petros B, Polderman AM, 2004. The use of real-time PCR to identify E. histolytica and E. dispar infections in prisoners and primary-school children in Ethiopia. Ann Trop Med Parasitol 98: 4348.

    • Search Google Scholar
    • Export Citation
  • 27.

    Ayed SB, Abdallah RB, Mousli M, Aoun K, Thellier M, Bouratbine A, 2008. Molecular differentiation of E. histolytica and E. dispar from Tunisian food handlers with amoeba infection initially diagnosed by microscopy. Parasite 15: 6568.

    • Search Google Scholar
    • Export Citation
  • 28.

    Santos FLN, de Souza Gonçalves M, Soares NM, 2011. Validation and utilization of PCR for differential diagnosis and prevalence determination of E. histolytica/E. dispar in Salvador City, Brazil. Braz J Infect Dis 15: 119125.

    • Search Google Scholar
    • Export Citation
  • 29.

    Opintan JA, Newman MJ, Ayeh-Kumi PF, Affrim R, Gepi-Attee R, Sevilleja JEAD, Roche JK, Nataro JP, Warren CA, Guerant RL, 2011. Pediatric diarrhea in southern Ghana: etiology and association with intestinal inflammation and malnutrition. Am J Trop Med Hyg 83: 936943.

    • Search Google Scholar
    • Export Citation
  • 30.

    Gupta SS, Singh O, Shukla S, Raj MK, 2009. Acute fulminant necrotizing amoebic colitis: a rare and fatal complication of amebiasis: a case report. Cases J 2: 65576561.

    • Search Google Scholar
    • Export Citation
  • 31.

    Wanke C, Butler T, Islam M, 1988. Epidemiologic and clinical features of invasive amebiasis in Bangladesh: a case-control comparison with other diarrheal diseases and postmortem findings. Am J Trop Med Hyg 38: 335341.

    • Search Google Scholar
    • Export Citation
  • 32.

    Roy S, Kabir M, Mondal D, Ali IK, Petri WA Jr, Haque R, 2005. Real-time-PCR assay for diagnosis of Entamoeba histolytica infection. Clin Microbiol 43: 21682172.

    • Search Google Scholar
    • Export Citation
  • 33.

    Visser LG, Verweij JJ, Van Esbroeck M, Edeling WM, Clerinx J, Polderman AM, 2006. Diagnostic methods for differentiation of Entamoeba histolytica and Entamoeba dispar in carriers: performance and clinical implications in a non-endemic setting. Int J Med Microbiol 296: 397403.

    • Search Google Scholar
    • Export Citation
  • 34.

    Cegielski JP, Ortega YR, McKee S, Madden JF, Gaido L, Schwartz DA, Manji K, Jorgensen AF, Miller SE, Pulipaka UP, Msengi AE, Mwakyusa DH, Sterling CR, Reller LB, 1999. Cryptosporidium, Enterocytozoon, and Cyclospora infections in pediatric and adult patients with diarrhea in Tanzania. Clin Infect Dis 28: 314321.

    • Search Google Scholar
    • Export Citation
  • 35.

    Tumwine JK, Kekitiinwa A, Bakeera-Kitaka S, Ndeezi G, Downing R, Feng X, Akiyoshi DE, Tzipori S, 2005. Cryptosporidiosis and microsporidiosis in Ugandan children with persistent diarrhea with and without concurrent infection with the human immunodeficiency virus. Am J Trop Med Hyg 73: 921925.

    • Search Google Scholar
    • Export Citation
  • 36.

    Houpt ER, Bushen OY, Sam NE, Kohli A, Asgharpour A, Ng CT, Calfee DP, Guerrant RL, Maro V, Ole-Nguyaine S, Shao JF, 2005. Short report: asymptomatic Cryptosporidium hominis infection among human immunodeficiency virus-infected patients in Tanzania. Am J Trop Med Hyg 73: 520522.

    • Search Google Scholar
    • Export Citation
  • 37.

    Esteban JG, Aguirre C, Flores A, Strauss W, Angles R, Mas-Coma S, 1998. High Cryptosporidium prevalences in healthy Aymara children from the northern Bolivian Altiplano. Am J Trop Med Hyg 58: 5055.

    • Search Google Scholar
    • Export Citation
  • 38.

    Yu JR, Lee JK, Seo M, Kim SI, Sohn WM, Huh S, Choi HY, Kim TS, 2004. Prevalence of cryptosporidiosis among the villagers and domestic animals in several rural areas of Korea. Korean J Parasitol 42: 16.

    • Search Google Scholar
    • Export Citation
  • 39.

    Abba K, Sinfield R, Hart CA, Garner P, 2009. Pathogens associated with persistent diarrhoea in children in low and middle income countries: systematic review. BMC Infect Dis 9: 88103.

    • Search Google Scholar
    • Export Citation
  • 40.

    Uneke C, Nnachi M, Aruna U, 2008. Assessment of polyparasitism with intestinal parasite infections and urinary schistosomiasis among school children in a semi-urban area of southeastern Nigeria. Internet J Health 9: 1. Available at: https://ispub.com/IJH/9/1/8481.

    • Search Google Scholar
    • Export Citation
  • 41.

    Ukpai OM, Ugwa CD, 2003. The prevalence of gastro-intestinal tract parasites in primary school children in Ikwuano Local Government Area of Abia State, Nigeria. Nig J Parasitol 24: 129136.

    • Search Google Scholar
    • Export Citation
  • 42.

    Wegayehu T, Adamu H, Petros B, 2013. Prevalence of Giardia duodenalis and Cryptosporidium species infections among children and cattle in north Shewa Zone, Ethiopia. BMC Infect Dis 13: 419426.

    • Search Google Scholar
    • Export Citation
  • 43.

    Van Lint P, Rossen JW, Vermeiren S, Ver Elst K, Weekx S, Van Schaeren J, Jeurissen A, 2013. Detection of Giardia lamblia, Cryptosporidium spp. and Entamoeba histolytica in clinical stool samples by using multiplex real-time PCR after automated DNA isolation. Acta Clin Belg 68: 188192.

    • Search Google Scholar
    • Export Citation

 

 

 

 

Molecular Detection of the Carriage Rate of Four Intestinal Protozoa with Real-Time Polymerase Chain Reaction: Possible Overdiagnosis of Entamoeba histolytica in Nigeria

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  • Institute of Medical Microbiology and Infectious Disease Epidemiology, Medical Faculty, University of Leipzig, Leipzig, Germany; Ebonyi State University, Abakaliki, Nigeria; Department of Clinical Microbiology, Centre for Medical Parasitology, Copenhagen University Hospital Copenhagen, Denmark; Department of International Health, Immunology, and Microbiology, University of Copenhagen, Denmark; Department of Microbiology and Infection Control, Statens Serum Institut, Copenhagen, Denmark

Diarrhea remains the second largest killer of children worldwide, and Nigeria ranks number two on the list of global deaths attributable to diarrhea. Meanwhile, prevalence studies on potentially diarrheagenic protozoa in asymptomatic carriers using molecular detection methods remain scarce in sub-Saharan countries. To overcome sensitivity issues related to microscopic detection and identification of cysts in stool concentrates, real-time polymerase chain reaction (PCR) was used to analyze genomic DNAs extracted from stool samples from 199 healthy school children for Entamoeba histolytica, E. dispar, Giardia intestinalis, and Cryptosporidium. Questionnaires were administered for epidemiological data collection. E. histolytica was not detected in any of the samples, whereas Giardia (37.2%), E. dispar (18.6%), and Cryptosporidium (1%) were found. Most of the children sourced their drinking water from community wells (91%), while the majority disposed of feces in the bush (81.9%). Our study is the first to use real-time PCR to evaluate the epidemiology of E. histolytica, Giardia, and Cryptosporidium in Nigeria where previous studies using traditional diagnostic techniques have suggested higher and lower carriage rates of E. histolytica and Giardia, respectively. It is also the first study to accurately identify the prevalence of common potentially diarrheagenic protozoa in asymptomatic carriers in sub-Saharan Africa.

Introduction

Diarrhea is the second leading cause of death among children aged less than 5 years, globally causing about 1.5 million deaths each year. The disease kills more young children than acquired immunodeficiency syndrome (AIDS), malaria, and measles combined.1 Africa and south Asia bear more than 80% of the global share of child deaths because of diarrhea, while country-wise, Nigeria with 151,700 diarrhea-related fatalities per year, is currently ranked number two, topped only by India.2 Apart from direct mortality, persistent diarrhea has been associated with malnutrition, poor growth, as well as systemic infections of the respiratory and urinary tracts.3,4 Although intestinal protozoal infections because of Entamoeba histolytica and Giardia are among the wide range of pathogens responsible for diarrhea, rotavirus, Cryptosporidium, and bacterial agents such as Escherichia coli and Shigella are the most common etiologic agents.5 A recent survey of data on childhood deaths collected between 2000 and 2012 in seven low- and middle-income countries showed that persistent diarrhea requiring investigation accounted for more than 30% of diarrheal deaths in infants aged 1–11 months at 5/7 sites and more than 25% of deaths in children aged 1–4 years at 3/5 sites for which data were available. It was further revealed that at six of the seven study sites, 70–100% of children with persistent diarrhea who died had been seen in a health-care facility, while more than 50% of them had received oral rehydration solutions or intravenous fluids.6

Protozoa are important causes of persistent diarrhea,2 and current emphasis on rehydration fluid is adequate only for acute diarrhea agents such as rotavirus, which is self-limiting if the lost fluid and electrolytes are adequately replaced. These observations raise fundamental questions as to the quality of diagnosis and care provided to these children.

Apart from overall issues with timely and accurate diagnosis of intestinal protozoal infections in sub-Saharan Africa, the lack of ability to differentiate between E. histolytica and nonpathogenic amoebic species has erroneously inflated the etiologic role of the former as a diarrhea-causing agent,7 potentially leading to mismanagement of diarrheal disease, while the real causative agents may continue to spread undeterred,8 with the additional risk of emergence of organisms that are antimicrobially resistant.9 Organism-specific stool testing is also important for public health reasons because results can serve as early warning of an impending outbreak.10

The diagnosis of E. histolytica infection has traditionally relied on microscopic examination of fresh or fixed stool specimens.11 Meanwhile, microscopy has at least two major limitations; one is its suboptimal sensitivity, which is about 60%,12 while the second is its inability to distinguish potentially pathogenic E. histolytica from morphologically identical but nonpathogenic E. dispar, E. moshkovskii, and other quadrinucleate cysts of Entamoeba.

Serological methods have become useful for detection of E. histolytica infections in developed countries. However, serological tests are unable to definitively distinguish past from current infections in developing countries where amoebiasis is more common. Some of the serological tests are either costly, time consuming, require special skills, insensitive, or unspecific, or suffer from a combination of these setbacks.13

Newer approaches, including polymerase chain reaction (PCR) and antigen-based enzyme-linked immunosorbent assays (ELISAs), have emerged as means of solving the challenge of detecting E. histolytica.12

Several studies have been carried out to compare the performance of the newer methods, showing that PCR has better sensitivity and specificity in the detection of E. histolytica.12,1416

Strongly endorsed by the World Health Organization (WHO),17 the use of PCR is currently the method of choice for clinical and epidemiological studies of amebiasis in developed countries. However, the problem of overdiagnosis of E. histolytica remains a major problem in developing countries despite the fact that species-specific PCR methods have been available for more than a decade. The use of laboratory methods not enabling differentiation between E. histolytica and nonpathogenic amoebae has largely been responsible for uncertainty regarding the epidemiology of amebiasis as well as the frequently quoted global prevalence rate of this pathogen.15

The epidemiology of E. histolytica infections in Nigeria hence remains unknown because nonmolecular methods are still being used in investigations, with prevalence figures ranging from 35.4% to 72%,18,19 typically around 40% as estimated by one of the earliest studies some 25 years ago.20

There are very few studies on the prevalence of intestinal protozoa in asymptomatic carriers. Such studies are critical to gaining an understanding of the clinical and public health significance of intestinal protozoa. To the best of our knowledge, this study is the first attempt to establish the epidemiology of the prevalence of not only E. histolytica, but also E. dispar, Giardia, and Cryptosporidium in healthy children in Nigeria using real-time PCR.

Materials and Methods

Study site and study population.

Sample collection was carried out in the town of Ilero (latitude, 8°4′ 0N; longitude, 3°21′ 0E), a semirural community in southwestern Nigeria. The vegetation of Ilero is Guinea savanna with a distinct rainy season from April to September and a dry season from October to March. Farming is the predominant occupation of the inhabitants of this community. The town has a population of about 35,000 inhabitants.21 All the schools also run preschool nursery facilities, thereby giving us the opportunity to sample children below the age of 6 years as well. There were seven primary schools in this community where stool samples were collected during a mass deworming program. About 1,442 pupils submitted stool samples from which 199 were randomly selected for DNA extraction and PCR-based screening for selected intestinal protozoa.

Questionnaires.

Questionnaires were administered to children and their parents/guardians to obtain information about sociodemographic data such as age, religion, ethnic group, father's occupation, source of drinking water, means of feces disposal, and so on. The information obtained was entered into an Excel spreadsheet for future analysis.

DNA extraction.

The QIAamp DNA Stool Mini Kit (Qiagen GmbH, Hilden, Germany) was used to extract DNA from 200 mg frozen stool samples according to the manufacturer's instructions but with some modifications. The modification carried out involved the addition of about 0.3 g zirconium-silica beads (diameter, 0.1 mm; Biospec Product Inc., Bartlesville, OK) to each tube just before the first heating step at 95°C for 5 minutes, after which the sample was shaken at 30 Hz for 6 minutes by a TissueLyser (QiagenRetsch GmbH, Hanover, Germany). This modification was carried out to increase DNA yield for downstream processing.22

Real-time PCR.

Real-time PCR for Giardia, E. histolytica, and E. dispar was performed using the primers and protocols described by Verweij and others.23,24 For Cryptosporidium real-time PCR, an in-house protocol (Stensvold, unpublished) was used for the amplification of the small subunit (SSU) rRNA gene of the genus Cryptosporidium.

Positive and negative controls were included in each real-time PCR, run as part of the standard protocol of the Laboratory of Parasitology, Statens Serum Institut, Copenhagen, Denmark. Furthermore, the real-time assays had previously been validated by spiking assays (sensitivity) and confirmatory PCR and sequencing of positive samples (specificity).

To corroborate our real-time PCR findings, 50 of the samples were also tested using an alternative method. The TechLab E. histolytica II kit (Techlab, Blacksburg, VA)—a second generation monoclonal ELISA-based test for detecting E. histolytica galactose adhesin in fecal samples—was used for E. histolytica. The test was performed according to the manufacturer's instructions. ELISA results were analyzed by an automatic microtiter plate reader (Anthosht III; AnthosLabtec, Germany) at 450 nm. Positive results were defined as an optical density reading of ≥ 0.05 after subtraction of the negative control optical density.

For both Cryptosporidium and Giardia, a direct immunofluorescence (DIF) microscopy test, Crypto/Giardia Cel, (Cellabs Pty Ltd., Brookvale, New South Wales, Australia) was used. The tests were carried out in line with the manufacturer's instructions, incorporating positive and negative controls into each analysis.

Ethical issues.

Ethical approval for the study was issued by the research and ethical committee of the Federal Teaching Hospital, Abakaliki, Nigeria. Written informed consent was also obtained from all the parents or guardians of the participating pupils. Parents and guardians were told that participation in our study was voluntary and all participating children were given free deworming tablets irrespective of stool sample submission.

Statistical analysis.

Data were entered into the EPI-info (3.5.3) statistical software. Descriptive statistics were used to cross-tabulate the variables. The χ2 test with Yate's correction was used for comparison of categorical data and Student's t test was used for comparison of normally distributed data. A P value < 0.05 was considered statistically significant.

Results

Most of our study subjects were aged 6–10 years (61.3%) followed by those who were 5 years or less (Table 1). The majority of the children (90.5%) were from the Yoruba ethnic group.

Table 1

Distribution of intestinal protozoa in relation to sociodemographic characters of the study population

CharacteristicEntamoeba histolytica N (%)E. dispar N (%)Giardia N (%)Cryptosporidium N (%)
Age (years)
 < 6, N = 4603 (6.5)19 (41.3)0
 6–10, N = 122027 (22.1)43 (35.6)2 (1.6)
 11–14, N = 3107 (22.5)12 (38.7)0
Sex
 Male, N = 110019 (17.3)40 (36.4)1 (0.9)
 Female, N = 89018 (20.2)34 (38.2)1 (1.1)
Religion
 Christianity, N = 114022 (19.3)40 (35.1)1 (0.9)
 Islam, N = 85015 (17.5)34 (40.0)1 (1.1)
Tribe
 Yoruba, N = 180033 (18.3)66 (36.7)2 (1.1)
 Hausa, N = 60000
 Ibo, N = 1003 (30.0)7 (70.0)0
 Fulani, N = 301 (33.3)1 (33.3)0
Occupation
 Farming, N = 102019 (18.7)38 (37.3)2 (2.0)
 Trading, N = 3507 (20.0)18 (51.4)0
 Artisan, N = 2908 (27.6)9 (31.0)0
 Others, N = 2803 (20.0)9 (32.1)0
 Nil, N = 50000
Toilet facility
 Pit latrine, N = 3004 (13.3)11 (36.7)1 (3.3)
 Water closet, N = 601 (16.7)1 (16.7)0
 Bush, N = 162032 (19.8)62 (38.0)1 (0.6)
Drinking water
 Well, N = 181033 (18.2)68 (37.6)2 (1.1)
 Nylon sachet water N = 1403 (21.4)4 (28.6)0
 Stream N = 401 (25.0)2 (50.0)0

Of the study subjects, 114 (57.3%) were from Christian homes, and 55.3% were males.

Analysis of the data pertaining to the occupation of the fathers of these children showed that farming (51.2%) was the most common occupation in this community followed by trading (17.6%). Community wells (91%) represented the most common source of drinking water followed by water sealed in nylon sachets (7.0%). Tap water was not available in the community. The major form of sewage disposal was in the bush (81.4%) followed by the use of pit latrines (15.1%).

No DNA of E. histolytica was detected in any of the stool samples, while E. dispar was identified in the stool of 37 (18.6%) children by real-time PCR. Giardia, detected in 74 (37.2%) of the children, was the most common intestinal protozoon identified, while only two (1.0%) cases of Cryptosporidium were observed (Table 1).

Giardia (41.3%) was more prevalent in children below the age of 6 years whereas E. dispar was more common in the older age groups. The two cases of Cryptosporidium occurred in children aged 6–10 years. Giardia was more common in females (38.2%) and children from the Ibo (70.0%) ethnic group. The two Cryptosporidium cases were seen in children from farming homes, while Giardia was most common in children whose fathers were traders (51.4%). E. dispar (19.8%) and Giardia (38.0%) were more common in children who disposed feces in the bush, while these two protozoa were also more prevalent in children who sourced their drinking water from the stream, (25% and 50%, respectively; Table 1). However, no statistically significant differences between any of the groups were found (P > 0.05).

Supplementary laboratory tests were performed on 50 selected samples (age groups < 6 years, N = 10; 6–10 years, N = 30; 11–14 years, N = 10). Of these 50 samples, two were PCR-positive for Cryptosporidium, 24 were PCR-positive for Giardia, and 30 were PCR-positive for E. dispar. All 50 samples were negative for Cryptosporidium by DIF microscopy, including the two samples that were previously positive by real-time PCR. All the samples that were real-time PCR-negative for Giardia were also negative by DIF microscopy, while 10/24 samples positive for Giardia by real-time PCR were positive by DIF. Hence, 14/24 real-time PCR-positive samples were negative by DIF microscopy for Giardia, yielding a DIF sensitivity of 41.67% compared with real-time PCR.

The mean cycle threshold (CT value) for the Giardia real-time PCR was 35.5 with a range of 28.6–44.0 and a standard deviation (SD) of 3.55, while the mean CT value for E. dispar was 35.5 with a range of 27.4–42.5 and an SD of 4.6.

The mean CT value for DIF-microscopy-positive samples was 31.5 (SD, 1.7), which was significantly lower than that for DIF-microscopy-negative samples (mean, 37.5 [SD, 2.5]; P < 0.001), suggesting that DIF-microscopy-positive samples had a higher parasite load compared with microscopy-negative samples.

Regarding E. histolytica, all 50 samples (comprising 30 E. dispar PCR-positive samples and 20 E. dispar PCR-negative samples) were negative by antigen ELISA, confirming PCR results and the low prevalence of this species in healthy children.

Discussion

Our findings showed a surprisingly low prevalence of E. histolytica carriage in Nigerian children compared with previous studies. Although the absence of E. histolytica in our study population was initially puzzling given the fact that majority of these children source their drinking water from community wells (91%) and also dispose their feces in bushes (81.4%), the high prevalence of Giardia (37.2%) showed that the lifestyle in this community still involves a high degree of transmission of intestinal pathogens. Various types of intestinal helminthes22 and a very high prevalence of Blastocystis (Efunshile and Stensvold, unpublished data) were also discovered in this community, which may indeed evince the mode of unhygienic fecal disposal and fecal–oral transmission. There was no significant association between the prevalence of giardiasis and any of the sociodemographic characters in our study population, but any such associations may be blurred by extensive overall risk behavior. The trend toward a higher prevalence of Giardia in younger children, suggesting a particular risk behavior in this group, was in agreement with observations from a Ugandan study.25

The low carriage rate of E. histolytica in our study is in contrast to previous studies in Nigeria where higher prevalence figures were reported.1820 These studies were all based on microscopic stool investigations that do not discriminate between E. dispar, which was also prevalent in our study, and E. histolytica. In addition, one of the studies focused on patients with diarrhea.18 Since we have used the most sensitive and specific diagnostic tool (PCR) recommended by the WHO, shown to be able to detect a single trophozoite of E. histolytica compared with diagnostic alternatives,15 at least we can say that there is no sign of a reservoir of asymptomatic carriage among children as evidenced by this study.

Our result is in agreement with molecular studies in many other developing countries, for example Ethiopia, where Kebede and others used real-time PCR to investigate 214 stool samples that were reportedly positive by microscopy for Entamoeba only to find that none of the samples were positive for E. histolytica DNA.26 Also, a molecular survey of stool samples from food handlers previously diagnosed with amebic infection by microscopy in Tunisia eventually showed that no one was infected by this species.27 In another study in Brazil, a molecular reevaluation of 227 stool samples microscopically positive for the E. histolytica/E. dispar complex showed that only E. dispar was present,28 and a recent study showed that E. histolytica was absent among a selected, apparently healthy, population of children in Ghana, contrary to previous belief.29 The availability of molecular methods to distinguish between E. dispar and E. histolytica has thus thrown into question the commonly accepted figure of 500 million E. histolytica infections worldwide,7 suggesting that the true global prevalence may have been largely overestimated.

It is important to correctly diagnose diarrheal patients not only to reduce the morbidity and mortality of amebiasis but also to minimize the undue treatment of patients infected with E. dispar and potentially other species of Entamoeba in general to better be able to match treatment with diarrhea-causing microorganisms. For instance, overdiagnosis of E. histolytica implies that colitis resulting from bacterial agents such as Campylobacter and Shigella species will probably be inappropriately treated with antiprotozoal drugs, while failure to specifically diagnose E. histolytica is associated with fatal consequences.30,31

There are still large gaps in the knowledge of species prevalence rates in different regions of the world, particularly in the African continent where very few studies are being performed using molecular methods. To address this deficit, there is a need to implement species-specific diagnosis of E. histolytica. Moreover, there is a need to strengthen collaboration between researchers from developing countries and those from developed countries so as to be able to effectively identify the true epidemiology of these parasites, and hopefully produce novel, accurate, sensitive, easy-to-use, inexpensive, sustainable, and highly applicable diagnostics that circumvents the requirement for advanced technological infrastructure.7 This will in turn improve rational use of antimicrobial agents and treatment outcomes. The TechLab II ELISA could be useful as a diagnostic tool to rule out E. histolytica in an area where nonpathogenic amoeba species such as E. dispar are endemic. The agreement between this method and real-time PCR in our study population is similar to the observation of the research group of Roy32 and that of Visser.33 However, because of the lack of positive results our study could not be used to validate this method.

The low prevalence of Cryptosporidium (1%) noted in this study was not surprising since this particular protozoon has been shown to be more prevalent in immunocompromised individuals and in cases of diarrhea.5 For instance, in Tanzania, Cegielski and others found a prevalence of 15% in human immunodeficiency virus (HIV)–infected children with diarrhea, while Cryptosporidium was not found among healthy controls.34

Similarly, among HIV-positive Ugandan children with diarrhea, a prevalence of 73.6% was reported compared with 5.9% among the controls. The Uganda study further showed that Cryptosporidium was essentially associated with low CD4 cell counts.35 Elsewhere, cryptosporidiosis has been shown to be a marker of low CD4 count.36 However, in contrast to our results, a high Cryptosporidium carriage rate was reported in community-based studies in Bolivia37 (31.6%) and Korea38 (3.3%) among healthy individuals. Over-crowding and direct contact with cattle was attributed to the high rate in their studies, but it should be noted that microscopy—not PCR—was used as the screening method in both studies.

The high prevalence of Giardia recorded in our study was similar to the findings of Kotloff and others in the recently published data from Global Enteric Multicenter Study (GEMS)5 where Giardia was surprisingly discovered to be significantly more prevalent in the control group of children without diarrhea than in the cases. A systematic review by Abba and others discovered that the various types of intestinal pathogens commonly associated with persistent diarrhea in children can also be asymptomatically carried with a similar frequency in children without diarrhea and came up with the recommendation that new research will be needed across countries to help map the causes and be able to explore effective options for empirical treatment.39 Previous studies based on traditional microscopy by Uneke and others (2.3%)40 and Ukpai and others (1.7%)41 in Nigeria and also by Wegayehu and others from Ethiopia (13.8%)42 have shown a lower carriage rate of Giardia. This led us to compare the real-time PCR result with another highly specific test for Giardia, that is, DIF microscopy. Using this technique, the estimated carriage rate dropped to 15.5% in our study population. The observation of fewer Giardia-positive samples and no Cryptosporiodium-positive samples by DIF microscopy compared with real-time PCR was not unexpected; a previous study showed PCR to be the more sensitive method for detecting these organisms.43

The high CT values in DIF-microscopy-negative cases indicate that PCR testing for Giardia may be too sensitive to discriminate between clinically relevant infection and asymptomatic carriage. Further studies should address this question and also clarify the potential importance of an asymptomatic carrier state.

ACKNOWLEDGMENTS

We thank the German Academic Exchange Services (DAAD) for providing the scholarship grant (Code number A/10/98666) that enabled this research. We also thank the staff of Baptist Medical Center, Ilero, Oyo state in Nigeria for their support during the field work. We also appreciate the teachers and pupils of all the public primary schools in Ilero, Nigeria, for participating voluntarily in this project.

  • 1.

    Liu L, Johnson HL, Cousens S, Perin J, Scott S, Lawn JE, Rudan I, Campbell H, Cibulskis R, Li M, Mathers C, Black RE; Child Health Epidemiology Reference Group of WHO and UNICEF, 2012. Global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since 2000. Lancet 379: 21512161.

    • Search Google Scholar
    • Export Citation
  • 2.

    UNICEF, WHO, 2009. Diarrhoea: Why Children are Still Dying and What Can Be Done. New York, NY: United Nations Children's Fund. Available at: http://www.who.int/child_adolescent_health/documents/who. Accessed October 6, 2014.

    • Search Google Scholar
    • Export Citation
  • 3.

    Das SK, Faruque AS, Chisti MJ, Malek MA, Salam MA, Sack DA, 2012. Changing trend of persistent diarrhoea in young children over two decades: observations from a large diarrheal disease hospital in Bangladesh. Acta Paediatr 101: e452e457.

    • Search Google Scholar
    • Export Citation
  • 4.

    World Health Organization and United Nations Children's Fund, 2013. Ending Preventable Child Deaths from Pneumonia and Diarrhea by 2025. The Integrated Global Action Plan for Pneumonia and Diarrhea (GAPPD). Geneva, Switzerland and New York, NY: World Health Organization and United Nations Children's Fund. Available at: http://apps.who.int/iris/bitstream/10665/79200/1/9789241505239_eng.pdf?ua=1. Accessed October 4, 2014.

    • Search Google Scholar
    • Export Citation
  • 5.

    Kotloff KL, Nataro JP, Blackwelder WC, Nasrin D, Farag TH, Panchalingam S, Wu Y, Sow SO, Sur D, Breiman RF, Faruque ASG, Zaidi AKM, Saha D, Alonso PL, Tamboura B, Sanogo D, Onwuchekwa U, Manna B, Ramamurthy T, Kanungo S, Ochieng JB, Omore R, Oundo JO, Hossain A, Das SK, Ahmed S, Qureshi S, Quadri F, Adegbola RA, Antonio M, Hossain MJ, Akinsola A, Mandomando I, Nhampossa T, Acácio S, Biswas K, O'Reilly CE, Mintz ED, Berkeley LY, Muhsen K, Sommerfelt H, Robins-Browne RM, Levine MM, 2013. Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study. Lancet 382: 209222.

    • Search Google Scholar
    • Export Citation
  • 6.

    Rahman AE, Moinuddin M, Molla M, Worku A, Hurt L, Kirkwood B, Mohan SB, Mazumder S, Bhutta Z, Raza F, Mrema S, Masanja H, Kadobera D, Waiswa P, Bahl R, Zangenbergh M, Muheh L, 2014. Childhood diarrheal deaths in seven low- and middle-income countries. Bull World Health Organ 92: 664671.

    • Search Google Scholar
    • Export Citation
  • 7.

    Samie A, El Bakri A, Ra'ed Abu Odeh R, 2012. Amoebiasis in the Tropics: Epidemiology and Pathogenesis. Available at: www.intechopen.com. Accessed October 10, 2014.

    • Search Google Scholar
    • Export Citation
  • 8.

    Guerrant RL, Gilder TV, Steiner TS, Thielman NM, Slutsker L, Tauxe RV, Hennessy T, Griffin PM, DuPont H, Sack RB, Tarr P, Neill M, Nachamkin I, Reller LB, Osterholm MT, Bennish ML, Pickering LK, 2001. Practice guidelines for the management of infectious diarrhea. CID 32: 231251.

    • Search Google Scholar
    • Export Citation
  • 9.

    Morris AJ, Murray PR, Reller LB, 1996. Contemporary testing for enteric pathogens: the potential for cost, time, and health care savings. J Clin Microbiol 34: 17761778.

    • Search Google Scholar
    • Export Citation
  • 10.

    Hines J, Nachamkin I, 1996. Effective use of the clinical microbiology laboratory for diagnosing diarrheal diseases. Clin Infect Dis 23: 12921301.

    • Search Google Scholar
    • Export Citation
  • 11.

    González-Ruiz A, Haque R, Aguirre A, Castañón G, Hall A, Guhl F, Ruiz-Palacios G, Miles MA, Warhurst DC, 1994. Value of microscopy in the diagnosis of dysentery associated with invasive Entamoeba histolytica. J Clin Pathol 47: 236239.

    • Search Google Scholar
    • Export Citation
  • 12.

    Tanyuksel M, Petri WA Jr, 2003. Laboratory diagnosis of amebiasis. Clin Microbiol Rev 16: 713729.

  • 13.

    Stark FRD, Beebe N, Marriott D, Elli J, Harkness J, 2007. A review of laboratory diagnostic techniques for Entamoeba species. Clin Microbiol Rev 20: 511532.

    • Search Google Scholar
    • Export Citation
  • 14.

    Haque R, Ali IK, Akther S, Petri WA Jr, 1998. Comparison of PCR, isoenzyme analysis, and antigen detection for diagnosis of E. histolytica infection. J Clin Microbiol 36: 449452.

    • Search Google Scholar
    • Export Citation
  • 15.

    Mirelman D, Nuchamowitz Y, Stolarsky T, 1997. Comparison of use of enzyme-linked immunosorbent assay-based kits and PCR amplification of rRNA genes for simultaneous detection of E. histolytica and E. dispar. J Clin Microbiol 35: 24052407.

    • Search Google Scholar
    • Export Citation
  • 16.

    Roy S, Kabir M, Mondal D, Ali IK, Petri WA Jr, Haque R, 2005. Real-time-PCR assay for diagnosis of E. histolytica infection. J Clin Microbiol 43: 21682172.

    • Search Google Scholar
    • Export Citation
  • 17.

    World Health Organization, 1997. Amebiasis. WHO Wkly Epidemiol Rec 72: 97100.

  • 18.

    Okeke IN, Ojo O, Lamikanra A, Kaper JB, 2003. Etiology of acute diarrhea in adults in southwestern Nigeria. J Clin Microbiol 41: 45254530.

  • 19.

    Idowu OA, Rowland SA, 2006. Oral fecal parasites and personal hygiene of food handlers in Abeokuta, Nigeria. Afr Health Sci 6: 160164.

  • 20.

    Oyerinde JPO, Alonge AA, Adegbite-Hollist AF, Ogunbi O, 1979. The epidemiology of Entamoeba histolytica in a Nigerian urban population. Int J Epidemiol 8: 5560.

    • Search Google Scholar
    • Export Citation
  • 21.

    Ilero Town in Nigeria. Available at: http://www.fallingrain.com/world/NI/00/Ilero.html. Accessed May 20, 2015.

  • 22.

    Smith B, Li N, Andersen AS, Slotved HC, Krogfelt KA, 2011. Optimising bacterial DNA extraction from faecal samples: comparison of three methods. Open Microbiol J 5: 1417.

    • Search Google Scholar
    • Export Citation
  • 23.

    Verweij JJ, Oostvogel F, Brienen EAT, Nang-Beifubah A, Ziem J, Polderman AM, 2003. Prevalence of E. histolytica and E. dispar in northern Ghana. Trop Med Int Health 8: 11531156.

    • Search Google Scholar
    • Export Citation
  • 24.

    Verweij JJ, Blangé RA, Templeton K, Schinkel J, Brienen EAT, van Rooyen MAA, van Lieshout L, Poldermanet AM, 2004. Simultaneous detection of E. histolytica, Giardia lamblia, and Cryptosporidium parvum in fecal samples by using multiplex real-time PCR. J Clin Microbiol 42: 12201223.

    • Search Google Scholar
    • Export Citation
  • 25.

    McElligott JT, Naaktgeboren C, Makuma-Massa H, Summer AP, Deal JL, 2013. Prevalence of intestinal protozoa in communities along the Lake Victoria region of Uganda. Int J Infect Dis 17: 658659.

    • Search Google Scholar
    • Export Citation
  • 26.

    Kebede A, Verweij JJ, Endeshaw T, Messele T, Tasew G, Petros B, Polderman AM, 2004. The use of real-time PCR to identify E. histolytica and E. dispar infections in prisoners and primary-school children in Ethiopia. Ann Trop Med Parasitol 98: 4348.

    • Search Google Scholar
    • Export Citation
  • 27.

    Ayed SB, Abdallah RB, Mousli M, Aoun K, Thellier M, Bouratbine A, 2008. Molecular differentiation of E. histolytica and E. dispar from Tunisian food handlers with amoeba infection initially diagnosed by microscopy. Parasite 15: 6568.

    • Search Google Scholar
    • Export Citation
  • 28.

    Santos FLN, de Souza Gonçalves M, Soares NM, 2011. Validation and utilization of PCR for differential diagnosis and prevalence determination of E. histolytica/E. dispar in Salvador City, Brazil. Braz J Infect Dis 15: 119125.

    • Search Google Scholar
    • Export Citation
  • 29.

    Opintan JA, Newman MJ, Ayeh-Kumi PF, Affrim R, Gepi-Attee R, Sevilleja JEAD, Roche JK, Nataro JP, Warren CA, Guerant RL, 2011. Pediatric diarrhea in southern Ghana: etiology and association with intestinal inflammation and malnutrition. Am J Trop Med Hyg 83: 936943.

    • Search Google Scholar
    • Export Citation
  • 30.

    Gupta SS, Singh O, Shukla S, Raj MK, 2009. Acute fulminant necrotizing amoebic colitis: a rare and fatal complication of amebiasis: a case report. Cases J 2: 65576561.

    • Search Google Scholar
    • Export Citation
  • 31.

    Wanke C, Butler T, Islam M, 1988. Epidemiologic and clinical features of invasive amebiasis in Bangladesh: a case-control comparison with other diarrheal diseases and postmortem findings. Am J Trop Med Hyg 38: 335341.

    • Search Google Scholar
    • Export Citation
  • 32.

    Roy S, Kabir M, Mondal D, Ali IK, Petri WA Jr, Haque R, 2005. Real-time-PCR assay for diagnosis of Entamoeba histolytica infection. Clin Microbiol 43: 21682172.

    • Search Google Scholar
    • Export Citation
  • 33.

    Visser LG, Verweij JJ, Van Esbroeck M, Edeling WM, Clerinx J, Polderman AM, 2006. Diagnostic methods for differentiation of Entamoeba histolytica and Entamoeba dispar in carriers: performance and clinical implications in a non-endemic setting. Int J Med Microbiol 296: 397403.

    • Search Google Scholar
    • Export Citation
  • 34.

    Cegielski JP, Ortega YR, McKee S, Madden JF, Gaido L, Schwartz DA, Manji K, Jorgensen AF, Miller SE, Pulipaka UP, Msengi AE, Mwakyusa DH, Sterling CR, Reller LB, 1999. Cryptosporidium, Enterocytozoon, and Cyclospora infections in pediatric and adult patients with diarrhea in Tanzania. Clin Infect Dis 28: 314321.

    • Search Google Scholar
    • Export Citation
  • 35.

    Tumwine JK, Kekitiinwa A, Bakeera-Kitaka S, Ndeezi G, Downing R, Feng X, Akiyoshi DE, Tzipori S, 2005. Cryptosporidiosis and microsporidiosis in Ugandan children with persistent diarrhea with and without concurrent infection with the human immunodeficiency virus. Am J Trop Med Hyg 73: 921925.

    • Search Google Scholar
    • Export Citation
  • 36.

    Houpt ER, Bushen OY, Sam NE, Kohli A, Asgharpour A, Ng CT, Calfee DP, Guerrant RL, Maro V, Ole-Nguyaine S, Shao JF, 2005. Short report: asymptomatic Cryptosporidium hominis infection among human immunodeficiency virus-infected patients in Tanzania. Am J Trop Med Hyg 73: 520522.

    • Search Google Scholar
    • Export Citation
  • 37.

    Esteban JG, Aguirre C, Flores A, Strauss W, Angles R, Mas-Coma S, 1998. High Cryptosporidium prevalences in healthy Aymara children from the northern Bolivian Altiplano. Am J Trop Med Hyg 58: 5055.

    • Search Google Scholar
    • Export Citation
  • 38.

    Yu JR, Lee JK, Seo M, Kim SI, Sohn WM, Huh S, Choi HY, Kim TS, 2004. Prevalence of cryptosporidiosis among the villagers and domestic animals in several rural areas of Korea. Korean J Parasitol 42: 16.

    • Search Google Scholar
    • Export Citation
  • 39.

    Abba K, Sinfield R, Hart CA, Garner P, 2009. Pathogens associated with persistent diarrhoea in children in low and middle income countries: systematic review. BMC Infect Dis 9: 88103.

    • Search Google Scholar
    • Export Citation
  • 40.

    Uneke C, Nnachi M, Aruna U, 2008. Assessment of polyparasitism with intestinal parasite infections and urinary schistosomiasis among school children in a semi-urban area of southeastern Nigeria. Internet J Health 9: 1. Available at: https://ispub.com/IJH/9/1/8481.

    • Search Google Scholar
    • Export Citation
  • 41.

    Ukpai OM, Ugwa CD, 2003. The prevalence of gastro-intestinal tract parasites in primary school children in Ikwuano Local Government Area of Abia State, Nigeria. Nig J Parasitol 24: 129136.

    • Search Google Scholar
    • Export Citation
  • 42.

    Wegayehu T, Adamu H, Petros B, 2013. Prevalence of Giardia duodenalis and Cryptosporidium species infections among children and cattle in north Shewa Zone, Ethiopia. BMC Infect Dis 13: 419426.

    • Search Google Scholar
    • Export Citation
  • 43.

    Van Lint P, Rossen JW, Vermeiren S, Ver Elst K, Weekx S, Van Schaeren J, Jeurissen A, 2013. Detection of Giardia lamblia, Cryptosporidium spp. and Entamoeba histolytica in clinical stool samples by using multiplex real-time PCR after automated DNA isolation. Acta Clin Belg 68: 188192.

    • Search Google Scholar
    • Export Citation

Author Notes

* Address correspondence to Michael A. Efunshile, Institute of Medical Microbiology and Infectious Disease Epidemiology, Faculty of Medicine, University of Leipzig, Liebigstraß 21, 04103 Leipzig, Germany. E-mail: drefunshile@yahoo.com

Authors' addresses: Michael A. Efunshile, Institute of Medical Microbiology and Infectious Disease Epidemiology, University of Leipzig, Leipzig, Germany, and Department of Medical Microbiology and Parasitology, Ebonyi State University, Abakaliki, Ebonyi, Nigeria, E-mail: drefunshile@yahoo.com. Bethrand A. F. Ngwu, Department of Medical Microbiology and Parasitology, Ebonyi State University, Abakaliki, Ebonyi, Nigeria, E-mail: bethfrancis5@yahoo.com. Jørgen A. L. Kurtzhals, Department of Clinical Microbiology, Centre for Medical Parasitology, Copenhagen University Hospital, Copenhagen, Denmark, and Department of International Health, Immunology, and Microbiology, University of Copenhagen, Copenhagen, Denmark, E-mail: joergen.kurtzhals@rh.regionh.dk. Sumrin Sahar, Department of Microbiology and Infection Control, Statens Serum Institute, Copenhagen, Denmark, E-mail: sumrinsahar@yahoo.com. Brigitte König, Institute of Medical Microbiology and Infectious Disease Epidemiology, University of Leipzig, Leipzig, Germany, E-mail: brigitte.koenig@medizin.uni-leipzig.de. Christen R. Stensvold, Department of Microbiology and Infection Control, Statens Serum Institut, Copenhagen, Denmark, E-mail: run@ssi.dk.

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