Of 260 children with acute diarrhea in Erbil, Iraqi Kurdistan, 96 (37%) were infected with rotavirus. Reverse transcription?polymerase chain reaction identified G1, G4, G2, G9, P[8], P[6], and P[4] as the most common genotypes. Eight G/P combinations were found, but P[8]G1 and P[4]G2 accounted for >50% of the strains

Ahmed Herish M.

Al-Rabaty Abas A.

Coulter J. Brian S.

Cunliffe Nigel A.

Dove Winifred

Hart C.A.

Nakagomi Osamu

Zaki Jamal M.

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MolecularCharacterization of RotavirusGastroenteritisStrains,Iraqi KurdistanHerish M. Ahmed,*†‡ J. Brian S. Coulter,* Osamu Nakagomi,†§ C. A. Hart,† Jamal M. Zaki,‡Abas A. Al-Rabaty,‡ Winifred Dove,† and Nigel A. Cunliffe†Of 260 children with acute diarrhea in Erbil, IraqiKurdistan, 96 (37%) were infected with rotavirus. Reversetranscription–polymerase chain reaction identified G1, G4,G2, G9, P[8], P[6], and P[4] as the most common geno-types. Eight G/P combinations were found, but P[8]G1 andP[4]G2 accounted for >50% of the strains.Rotavirus is the single most important cause of severegastroenteritis in young children throughout theworld. Globally, an estimated 702,000 children die eachyear due to rotavirus diarrhea (1). This large impact ofrotavirus disease has speeded the development of rotavirusvaccines, and 2 live, attenuated rotavirus vaccines areexpected to be available for global use within the next fewyears (1). Therefore, determining the prevalence and typesof rotaviruses within regions is essential to prepare forintroducing a vaccine.Rotavirus, a member of the family Reoviridae, has atriple-layered capsid that contains 11 segments of double-stranded genomic RNA. While protective immunityagainst rotavirus infection is not completely understood,serotype-specific immunity is believed to play a major role(1). Rotavirus serotypes are defined by genome segment 4for the P (protease-sensitive protein) type and by genomesegment 9 (or 7 or 8, depending on the strain) for the G(glycoprotein) type. Fourteen G types exist, of whichG1–G4 are commonly found in children with diarrhea, buta recent increase in the detection of serotype G8 and G9strains has captured considerable attention (2–4). While>24 P types have been reported in the literature, only P[4],P[6], and P[8] are commonly found among humanrotaviruses (1–3).In Iraq, the death rate in children <5 years of age wasreported to be 130/1,000 for boys and 120/1,000 for girlsin 2003 (5). Diarrhea is a major cause of illness and deathin Iraqi children; however, little information exists aboutthe origin of childhood diarrhea. Only a single studyshowed that rotavirus accounted for 24% of acute diarrheain hospitalized children in Basrah (6).The StudyThis descriptive, cross-sectional study of 6 weeks’duration was undertaken at Erbil Paediatric Hospital inIraqi Kurdistan between March and May 2005. The studyrecruited 260 children from 1 month to 5 years of age whowere admitted with acute diarrhea (defined as the passageof watery or loose stools >3 times per day for <2 weeks’duration). Basic demographic, epidemiologic, and clinicalinformation were collected prospectively, according to apro forma. Ethical approval for the research was obtainedfrom the review boards of the Liverpool School of TropicalMedicine and Erbil Paediatric Hospital. The hospitalserves a population of ≈1.5 million, and ≈3,116 births permonth occur in this population.A commercial enzyme-linked immunosorbent assay(ELISA) was used to detect rotavirus antigen (Rotaclone,Meridian Diagnostics, Cincinnati, OH, USA). Stool sam-ples were then stored frozen in the laboratory of the studyhospital until they were transported to Liverpool forrotavirus genotyping and electropherotyping. All samples(66) with an absorbance equal to or greater than the posi-tive control for the ELISA were subjected to genotyping.Rotavirus genomic RNA was extracted with guanidineisothiocyanate, followed by adsorption to and elution fromsilica particles according to the method described byGentsch et al. (7). The purified RNA was then used todetermine the P type and G type of rotavirus present in thestool specimens by reverse transcription-polymerase chainreaction as described by Gentsch et al. (7) and by Gouveaet al. (8). Rotavirus electropherotypes were determined bypolyacrylamide gel electrophoresis according to themethod described by Koshimura et al. (9), with some mod-ifications.Of 260 stool specimens tested by ELISA, 96 (37%)were positive for rotavirus. Rotavirus-positive patients hada mean age (SD) of 9.3 (8.5) months compared to 11.1(10.1) months in the rotavirus-negative patients. Theseresults suggest that rotavirus positive cases were slightlyyounger, although the difference was not statistically sig-nificant (p = 0.14). Rotavirus-positive patients were simi-lar to rotavirus-negative patients in most of the epidemio-logic and clinical characteristics (data not shown).However, rotavirus-positive patients were more likely toexhibit vomiting and have a shorter duration of diarrhea(p<0.01 for both analyses).DISPATCHES824 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 5, May 2006*Liverpool School of Tropical Medicine, Liverpool, UK; †Universityof Liverpool, Liverpool, UK; ‡Erbil Paediatric Hospital, Erbil, IraqiKurdistan; §Nagasaki University, Nagasaki, JapanOf the 66 rotavirus strains that underwent molecularcharacterization, 25 (38%) were G1, 11 (17%) were G2, 13(20%) were G4, and 7 (11%) were G9. Four (6%) weremixed infections (3 G1/G2, 1 G2/G4), and 6 (9%) were Gnontypeable. A total of 7 (11%) were P[4], 10 (15%) wereP[6], and 45 (68%) were P[8]. One showed mixed P[4] andP[8] genotypes (mixed with G1/G2), and 3 (5%) were Pnontypeable. None of the rotaviruses was both G and Pnontypeable.A total of 8 different P and G genotype combinationswere detected (Table). The most common combinationswere P[8]G1 (19, 33%), P[8]G4 (12, 21%), P[4]G2 (6,11%), P[6]G1 (6, 11%), and P[8]G9 (6, 11%). Theunusual combination of P[6]G9 was detected in 1 of thepatients.An electropherotype was obtained for 50 of the 66genotyped strains. Of these, 11 (22%) had a short electro-pherotype, and 39 (78%) had a long electropherotype(Table). Most of the short electropherotypes were theexpected G2 strains; however, 1 strain (P[8]G9) also had ashort electropherotype.ConclusionsThe only other study of viral gastroenteritis from Iraq(Basrah in the south) demonstrated that 24% of childrenwith acute gastroenteritis were infected with rotavirus (6).This figure is somewhat lower than the 37% detection ratein our study. Moreover, the prevalence we found is similarto those reported from neighboring countries such as Iran(35%) (10), Jordan (33%) (11), Kuwait (40%) (12), andTurkey (37%) (13). However, our study was undertakenover a 6-week period from the end of March to the begin-ning of May 2005. No information is available on the sea-sonal prevalence of rotavirus infection in Iraq, and a longerstudy is warranted to determine the true prevalence ofrotavirus infection and its seasonality in northern Iraq.However, the peaks of rotavirus infection in Iran, Kuwait,and Turkey were February–March, March–May, andDecember, respectively (10,12,13). More than 75% of ourcases of rotavirus diarrhea occurred in children <1 year ofage, with an overall mean age of slightly more than 9months. This pattern is similar to that in many developingcountries. In Jordan the mean age of children withrotavirus diarrhea was 7.2 months (10). However, in othercountries in the region the distribution was different; 30%of the infants with rotavirus in Iran were <1 year of age(10,12), 50% in Kuwait were <1 year of age,  and 63% inTurkey were <2 years of age (13). Although this study period was brief, we detected avariety of rotavirus strains. Four of the major globalhuman rotavirus genotypes (G1, G2, G4, G9) were detect-ed, as were each of the major P genotypes (P[4], P[6],P[8]). In Iran, in a study undertaken in 2001 and 2002, onlyG1 and G2 rotaviruses were detected, and the only P typeswere P[4] and P[8] (10), and in Turkey over a 2-year peri-od (2000–2002), G types G1–G4 and G9, as well as eachof the 3 major human P types were found (14). In Iraq, thecombinations P[8]G1 and P[8]G4 accounted for >50% ofthe strains of rotavirus. In Iran, P[8]G1 accounted for 95%of the strains, but P[8]G4 was not detected (10). In Turkey,P[8]G4 (42%) and P[8]G1 (27%) accounted for more thantwo thirds of the strains (14). G3 rotaviruses were notdetected in Iraq or Iran, and in Turkey only 1 of the 65strains was of genotype G3. Genotype G9 was detected in13% of the Iraqi strains, a similar finding to results inTurkey (14). We also detected mixed rotavirus infectionsin 6% of our patients, again similar to the findings inTurkey (14). The presence of mixed rotavirus infectionsindicates that new rotavirus strains may evolve by reassort-ment (1–3). Finally, among the G9 strains, one P[6]G9 had a longelectropherotype, and one P[8]G9 had a short electro-pherotype. The P[6]G9 and P[8]G9 strains were both cul-tured and subgrouped by ELISA with monoclonalantibodies and found to be of subgroup II. Partialsequences (831 bp) were obtained for their VP7 genes(AB247941 and AB247943; available from the DNA DataBank of Japan: www.ddbj.nig.ac.jp). They showed 99.4%similarity to each other and >99% similarity to strains fromAustralia (AY307087), Belgium (AY487858, AY487856),and India (RG9491165). A strain similar to our P[6]G9,called variant 3, was first detected in India, and strainssimilar to our P[8]G9, called variant 2, have beendescribed in Bangladesh and in the United States (15).Although the major global genotypes (except for G3strains) were detected, clearly, rotavirus strains are contin-uing to diversify in Iraq and other parts of the region. Thiscircumstance may pose challenges to the efficacy ofrotavirus vaccines.Rotavirus Gastroenteritis Strains, Iraqi KurdistanEmerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 5, May 2006 825AcknowledgmentsWe thank the Ministry of Health of the Kurdistan RegionalGovernment in Iraq for giving permission to undertake the studyand the management and staff of Erbil Paediatric Hospital fortheir cooperation and support.Dr Ahmed is a pediatrician who conducted this research aspart of the requirements for his master’s degree in tropical pedi-atrics. His research interests are in viral gastroenteritis and respi-ratory tract infections.References1. Cunliffe NA, Nakagomi O. A critical time for rotavirus vaccines.Expert Rev Vaccines. 2005;4:521–34.2. Santos N, Hoshino Y. Global distribution of rotavirus serotypes/geno-types and its implication for the development and implementation ofan effective rotavirus vaccine. Rev Med Virol. 2005;15:29–56.3. Gentsch JR, Laird AR, Bielfelt B, Griffin DD, Banyai K,Ramachandran M, et al. Serotype diversity and reassortment betweenhuman and animal rotavirus strains: implications for rotavirus vac-cine programs. J Infect Dis. 2005;192(Suppl 1):S146–59.4. Cunliffe NA, Dove W, Bunn JE, Ben Ramadan M, Nyangao JW,Riveron RL, et al. Expanding global distribution of rotavirus G9serotype: detection in Libya, Kenya, and Cuba. Emerg Infect Dis.2001;7:890–2.5. World Health Organization—Iraq. Statistics by country or region.2003 [cited 2005 Oct 19]. Available from http://www3.who.int/who-sis/country/indicators.cfm?country=IRQ&language=English. 6. Mahmood DA, Feachem RG. Clinical and epidemiological character-istics of rotavirus- and EPEC-associated hospitalized infantile diar-rhoea in Basrah, Iraq. J Trop Pediatr. 1987;33:319–25.7. Gentsch JR, Glass RI, Woods P, Gouvea V, Gorziglia M, Flores J, etal. Identification of group A rotavirus gene 4 types by polymerasechain reaction. J Clin Microbiol. 1992;30:1365–73.8. Gouvea V, Glass RI, Woods P, Taniguchi K, Clark HF, Forrester B, etal. Polymerase chain reaction amplification and typing of rotavirusnucleic acid from stool specimens. J Clin Microbiol. 1990;28:276–82.9. Koshimura Y, Nakagomi T, Nakagomi O. The relative frequencies ofG serotypes of rotaviruses recovered from hospitalized children withdiarrhoea: a 10 year survey (1987–1996) in Japan with a review ofglobally collected data. Microbiol Immunol. 2000;44:499–510.10. Khalili B, Cuevas LE, Reisi N, Dove W, Cunliffe NA, Hart CA.Epidemiology of rotavirus diarrhoea in Iranian children. J Med Virol.2004;73:309–12.11. Youssef M, Shurman A, Bougnoux M-E, Rawashdeh M, Bretagne S,Strockbine N. Bacterial, viral and parasitic enteric pathogens associ-ated with acute diarrhea in hospitalized children from northernJordan. FEMS Immunol Med Microbiol. 2000;28:257–63.12. Sethi SK, Al-Nakib W, Khuffash FA, Majeed HA. Acute diarrhea androtavirus infections in young children in Kuwait. Ann Trop Paediatr.1984;4:117–21.13. Karadag A, Acikgoz ZC, Avci Z, Catal F, Gocer S, Gamberzade S, etal. Childhood diarrhoea in Ankara, Turkey: epidemiological and clin-ical features of rotavirus-positive versus rotavirus negative cases.Scand J Infect Dis. 2005;37:269–75.14. Cataloluk O, Iturriza M, Gray J. Molecular characterization ofrotaviruses circulating in the population in Turkey. Epidemiol Infect.2005;133:673–8.15. Ramachandran M, Kirkwood CD, Unicomb L, Cunliffe NA, WardRL, Bhan MK, et al. Molecular characterization of serotype G9rotavirus strains from a global collection. Virology. 2000;278:436–44.Address for correspondence: C.A. Hart, Department of MedicalMicrobiology and Genitourinary Medicine, University of Liverpool,Duncan Building, Daulby St, Liverpool, L69 3GA, UK; email:cahmm@liverpool.ac.ukDISPATCHES826 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 5, May 2006Searchpast issues

Molecular characterization of rotavirus gastroenteritis strains, Iraqi Kurdistan

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Molecular characterization of rotavirus gastroenteritis strains, Iraqi Kurdistan

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