A new species of parvovirus tentatively named human bocavirus 4 (HBoV4) was genetically characterized. Among 641 feces samples from children and adults the most commonly detected bocaviruses species were HBoV2>HBoV3>HBoV4>HBoV1 with HBoV2 prevalence of 21% and 26% in Nigerian and Tunisian children. HBoV3 and HBoV4 species combined were found in 12/192 cases of non-polio acute flaccid paralysis (AFP) from Tunisia and Nigeria and 0/96 healthy Tunisian contacts (p=0.01). Evidence of extensive recombination at the NP1 and VP1 gene boundary between and within species was found. The multiple species and high degree of genetic diversity seen among the human bocaviruses found in feces relative to the highly homogeneous HBoV1 suggest that this world-wide distributed respiratory pathogen may have recently evolved from an enteric bocavirus, perhaps after acquiring an expanded tropism favoring the respiratory track. Elucidating the possible role of the newly identified enteric bocaviruses in human diseases including AFP and diarrhea will require further epidemiological studies.

Shigella spp. are exquisitely fastidious gram-negative organisms which frequently escape detection by traditional culture methods. To get a more complete understanding of the disease burden caused by Shigella in Nha Trang, Vietnam, real-time PCR was used to detect Shigella DNA. Randomly selected rectal swab specimens from 60 Shigella culture-positive patients and 500 Shigella culture-negative patients detected by populationbased surveillance of patients seeking care for diarrhea were processed by real-time PCR. The target of the primer pair is the invasion plasmid antigen H gene sequence (ipaH), carried by all four Shigella species and enteroinvasive Escherichia coli. Shigella spp. could be isolated from the rectal swabs of 547 of 19,206 (3%) patients with diarrhea. IpaH was detected in 55 of 60 (93%) Shigella culture-positive specimens, whereas it was detected in 87 of 245 (36%) culture-negative patients free of dysentery (P < 0.001). The number of PCR cycles required to detect a PCR product was highest for culture-negative, nonbloody diarrheal specimens (mean number of cycles to detection, 36.6) and was lowest for children with culture-positive, bloody diarrheal specimens (mean number of cycles, 25.3) (P < 0.001). The data from real-time PCR amplification indicate that the culture-proven prevalence of Shigella among patients with diarrhea may underestimate the prevalence of Shigella infections. The clinical presentation of shigellosis may be directly related to the bacterial load.

A coccidian organism assigned to the genus Cyclospora has been increasingly recognized in association with prolonged diarrhea in humans throughout the world. Confusion surrounds the taxonomy of this fastidious organism, despite the availability of morphology and sporulation characteristics. The small subunit rRNA coding region from cyclosporan oocysts purified from a human fecal specimen was amplified and sequenced. The same sequence was present in specimens from 8 other patients with cyclosporan oocysts but absent in specimens from asymptomatic subjects and from cryptosporidiosis patients. Phylogenetic analysis of rDNA sequences reveals that the human-associated Cyclospora is closely related to members of the Eimeria genus. These results allow predictions concerning Cyclospora host specificity, life cycle, and epidemiology as well as the development of a specific polymerase chain reaction-based diagnostic assay.

Detection of Shigella organisms and enteroinvasive Escherichia coli (EIEC) by polymerase chain reaction (PCR) was evaluated in 20 patients with dysentery before and in 17 of the 20 after treatment with ciprofloxacin. DNA sequences coding for IpaH antigen, a multiple copy sequence found on the chromosome, and the invasion plasmid locus (ial) was detected after DNA amplification in 13 stools from patients from whom shigellae or EIEC were isolated but not in 21 nondysenteric stools containing other enteric bacteria. Although shigellae or EIEC were not isolated from any patient with dysentery after ciprofloxacin treatment, IpaH and ial sequences were found after PCR amplification in 7 patients after treatment with ciprofloxacin. IpaH sequences alone were detected in 4 patients; DNA augmentation of IpaH in stools in a specific way to identify Shigella or EIEC infection in persons from whom cultures cannot be obtained promptly after the onset of diarrhea or who have received antibiotics.

Shigella spp. were isolated from 44%, Pleisomonas shigelloides from 22%, Campylobacter spp. from 16%, and Salmonella spp. from 10% of 200 Thai children with mucoid or bloody diarrhea. Enteroinvasive Escherichia coli (EIEC) was identified by examining isolates of E. coli for plasmids larger than 120 megadaltons (MDa), by identifying E. coli with a virulence marker antigen in an ELISA, and by performing DNA hybridization with a 17-kilobase (kb) EcoRI digestion fragment of plasmid pWR100 (a 140-MDa plasmid of Shigella flexneri 5). Sixty-four isolates of EIEC from 10 (5%) of the children were identified by these methods and confirmed by the Sereny test. All isolates of EIEC fermented lactose, and isolates of EIEC from seven children belonged to recognized serotypes of EIEC (O28ac:H- and O29:H-), whereas isolates of EIEC from three children were untypable. Examination of DNA from the total fecal growth with the 17-kb probe identified 9 of 10 children from whom EIEC were isolated and 2 of 102 children from whom EIEC or Shigella spp. were not isolated. Detection of EIEC by ELISA and DNA hybridization are important advances in defining the etiology of dysentery.