A short report on highlights of world-wide development of RIX4414: a Latin American experience.

An oral, human-derived monovalent (G1P1A) rotavirus vaccine, strain RIX4414, has been developed by GlaxoSmithKline, Rixensart, Belgium. The safety, immunogenicity and efficacy of this vaccine were evaluated in a randomized, double-blind, placebo-controlled, phase IIb trial conducted in Brazil, Mexico and Venezuela. Healthy infants were given two doses of vaccine (104.7, 105.2 or 105.8 ffu) or placebo at age 2 and 4 months, with routine DTPw-HBV and Hib vaccines. OPV was given separately, at least 2 weeks before or after administration of the study vaccine. A total of 2155 infants were enrolled, of whom 1618 received one of the three vaccine viral concentrations and 537 were given placebo. Analysis of efficacy included diarrheal episodes occurring from 2 weeks after second dose until one year of age. Efficacy rates against any rotavirus gastroenteritis, severe rotavirus gastroenteritis and hospitalizations for rotavirus disease were as high as 70% (46-84%; 95%CI), 86% (63-96%; 95%CI), and 93% (54-100%; 95%CI), respectively. For non-G1 (mainly G9) serotypes, RIX4414 vaccine conferred protection as high as 83% (40-97%; 95%CI) against severe gastroenteritis. A decrease was noted in the incidence of severe rotavirus-related gastroenteritis after first dose. It is demonstrated that two doses of RIX4414 are highly efficacious against severe rotavirus gastroenteritis and hospitalization, including disease caused by non-G1 strains, namely G9 serotypes.

Genetic diversity among sapoviruses.

Norovirus and Sapovirus are two genera of the family Caliciviridae that contain viruses that can cause acute gastroenteritis in humans. Noroviruses (NOR) are genetically highly diverse but limited studies of the genetic diversity of sapoviruses (SAP) have been reported. In this study we characterized twenty-five SAP detected in our laboratory from outbreaks or sporadic cases of acute gastroenteritis in children from different geographical locations and in adults involved in a cruise ship outbreak investigation and a nursing home outbreak. Based on significant differences of partial RNA polymerase sequences (278-286 nt), the 25 strains were grouped into 12 genetic clusters, including 9 potential new clusters. Extended sequence analysis of the capsid gene of selected strains representing five potential new clusters supported this grouping. Four strains (Hou7-1181/90, Mex340/90, Cruise ship/00 and Argentina39) had <84% amino acid (aa) identity to each other and to the published sequences in the GenBank. Mex14917/00 was almost identical to Stockholm/97/SE whose RNA polymerase sequence was unknown. Phylogenetic and distance analyses of the capsid region of the four new strains showed that Hou7-1181/90 and Argentina39 represent two new genogroups and Mex340/90 and Cruise ship/00 belong to two new clusters within the London/92 genogroup. Thus, based on the capsid sequences we propose to classify the currently known SAP into nine genetic clusters within five genogroups, including one genogroup that is represented by an animal calicivirus, the porcine enteric calicivirus (PEC).

Unexpected detection of animal VP7 genes among common rotavirus strains isolated from children in Mexico.

In the course of characterizing 103 rotaviruses from children in Mexico, we found that the majority of strains were globally common types (55.4% of total), while uncommon types represented 5.7%, mixed infections with common types represented 14.8%, and partially or fully nontypeable isolates represented about 24%. Serotype G9 was detected for the first time in Mexico. We sequenced a subset of strains that were G nontypeable by reverse transcriptase PCR and found surprisingly that two strains having common human rotavirus P genotypes (8 and 6) had serotype G3 and G4 VP7 gene sequences that shared closer homology with canine and porcine strains, respectively, than with human strains, suggesting that these isolates represented reassortants between human and animal rotaviruses.

rpoB Gene mutations in rifampin-resistant Mycobacterium tuberculosis identified by polymerase chain reaction single-stranded conformational polymorphism.

The use of polymerase chain reaction-single-stranded conformational polymorphism (PCR-SSCP) to study rpoB gene mutations in rifampin-resistant (RIFr) Mycobacterium tuberculosis has yielded contradictory results. To determine the sensitivity of this method, we analyzed 35 RIFr strains and 11 rifampin-susceptible (RIFs) strains, using the DNA sequencing of the core region of rpoB for comparison. Of the RIFr, 24 had a PCR-SSCP pattern identical to that of H37Rv; the other 11 had four different patterns. The 11 RIFs had PCR-SSCP patterns identical to that of H37Rv. The sensitivity of the assay was 31.4%; its specificity was 100%. We observed a strong correlation between the degree of resistance and the type of mutation.

Fucosylated human milk oligosaccharides vary between individuals and over the course of lactation.

Specific human milk oligosaccharides, especially fucosylated neutral oligosaccharides, protect infants against specific microbial pathogens. To study the concentrations of individual neutral oligosaccharides during lactation, a total of 84 milk samples were obtained from 12 women at 7 time periods during weeks 1-49 postpartum. The neutral oligosaccharides from each sample were isolated, perbenzoylated, resolved, and quantified by reversed-phase high-performance liquid chromatography. The resultant oligosaccharide peaks, identified by co-elution with authentic standards and mass spectrometry, ranged in size from tri- to octasaccharides. The total concentration of oligosaccharides declined over the course of lactation; the mean concentration at 1 year was less than half that in the first few weeks postpartum. One of the 12 donors produced milk fucosyloligosaccharides that were essentially devoid of alpha1,2 linkages (but contained alpha1,3- and alpha1,4-linked fucose) until late in lactation, consistent with the nonsecretor phenotype. In milk samples from the remaining 11 donors, fucosyloligosaccharides containing alpha1,2-linked fucose were prevalent, and their profiles were distinct from those of fucosyloligosaccharides devoid of alpha1,2-linked fucose. The ratio of alpha1,2-linked oligosaccharide concentrations to oligosaccharides devoid of alpha1,2-linked fucose changed during the first year of lactation from 5:1 to 1:1. Furthermore, the absolute and the relative concentrations of individual oligosaccharides varied substantially, both between individual donors and over the course of lactation for each individual. The patterns of milk oligosaccharides among individuals suggest the existence of many genotype subpopulations. This variation in individual oligosaccharide concentrations suggests that the protective activities of human milk could also vary among individuals and during lactation.

The risk of enteric infections associated with wastewater reuse: the effect of season and degree of storage of wastewater.

The effect of season and wastewater storage on the risk of Ascaris lumbricoides infection and diarrhoeal disease associated with wastewater reuse was studied in Mexico in 1991. Data were collected from 10,489 individuals during a dry-season survey. Exposure was to untreated wastewater, or effluent from 1 reservoir (< or = 1 nematode egg/L), or no wastewater irrigation (control group). The results were compared with a previous rainy-season survey which included effluent from 2 reservoirs in series. Direct exposure to untreated wastewater was associated with an excess risk of A. lumbricoides infection in children aged < 5 years (OR = 18.0) and persons aged > 5 years (OR = 13.5) and an increased risk of diarrhoea, particularly to children aged < 5 years (OR = 1.75); effects were stronger in the dry than in the rainy season. There was also an excess risk associated with the 1-reservoir group for A. lumbricoides infection (OR = 21.2 and 9.4) and for diarrhoeal disease (OR = 1.1 and 1.5) but little excess associated with the 2-reservoirs group. Therefore, wastewater retention in 1 reservoir (quality 10(5) faecal coliforms/100 mL, < or = 1 egg/L) does not significantly reduce risks of Ascaris infection and diarrhoeal disease whereas retention in 2 reservoirs in series (quality 10(3) faecal coliforms/100 mL, no detectable eggs/L) does.

Clinical manifestations and survival trends during the first 12 years of the AIDS epidemic in Mexico.

BACKGROUND: Our objective was to evaluate survival trends (1984-1995), the prevalence of AIDS-defining conditions, and the role of treatment with zidovudine and/or prophylaxis with trimethoprim-sulfamethoxazole (TMP-SMX) in survival following AIDS diagnosis. METHODS: We reviewed the clinical charts and postmortem studies of all patients admitted to the HIV Clinic from 1984-1995. Three groups were identified according to the following dates of HIV diagnosis: 1) 1984-1988; 2) 1989-1992, and 3) 1993-1995. RESULTS: We studied 909 charts. During the study period, 744 (81.6%) patients developed AIDS. Median survival increased from 11.7 months in group 1 to 15.4 and 17.5 months in groups 2 and 3, respectively (p <0.05). We observed the following important changes in the frequency of AIDS-defining conditions over the study period: Pneumocystis carinii pneumonia (PCP) decreased from 24.8 to 17 and 14% in groups 1, 2, and 3, respectively, (p = 0.008), and Kaposi's sarcoma (KS), from 31.1 to 10.5 and 13.5% (p <0.001). On the other hand, there was an increase in cytomegalovirus disease with 12.4, 20.4, and 18.6% (p = 0.04) and wasting syndrome with 36, 45, and 57% (p <0.001). In the proportional hazard model for death, zidovudine or TMP-SMX use was associated with a protective effect. CONCLUSIONS: Survival is improving among patients with HIV infection at our institution. The prevalence of AIDS-defining conditions has changed over the last 12 years. There has been a diminution of PCP and KS, whereas cases of CMV disease and wasting syndrome increased.

Molecular characterization of invasive and noninvasive Campylobacter jejuni and Campylobacter coli isolates.

Campylobacter jejuni is one of the most common causes of bacterial diarrhea worldwide and is the primary bacterial cause of food-borne illness. Adherence to and invasion of epithelial cells are the most important pathogenic mechanisms of Campylobacter diarrhea. Molecular characterization of invasive and noninvasive Campylobacter isolates from children with diarrhea and symptom-free children was performed by random amplified polymorphic DNA techniques (RAPD). A distinct RAPD profile with a DNA band of 1.6 kb was observed significantly more frequently among invasive (63%) than among noninvasive (16%) Campylobacter isolates (P = 0.000005). The 1.6-kb band was named the invasion-associated marker (IAM). Using specifically designed primers, a fragment of 518 bp of the iam locus was amplified in 85% of invasive and 20% of noninvasive strains (P = 0.0000000). Molecular typing with a PCR-restriction fragment length polymorphism assay which amplified the entire iam locus showed a HindIII restriction fragment polymorphism pattern associated mainly with invasive strains. Although cluster analysis of the RAPD fingerprinting showed genetic diversity among strains, two main clusters were identified. Cluster I comprised significantly more pathogenic and invasive isolates, while cluster II grouped the majority of nonpathogenic, noninvasive isolates. These data indicate that most of the invasive Campylobacter strains could be differentiated from noninvasive isolates by RAPD analysis and PCR using specific primers that amplify a fragment of the iam locus.

Molecular epidemiology of human astrovirus diarrhea among children from a periurban community of Mexico City.

Human astroviruses (HAstVs) were detected in 23 stool samples from 365 diarrhea episodes among 214 children (<18 months old) prospectively monitored for diarrhea in Mexico City. Stool samples were tested by EIA and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. EIA was less sensitive (74%) and equally specific, compared with RT-PCR analysis using type-common primers for HAstV detection. Of 31 HAstV isolates, EIA typed 18 (69%) of 26 EIA-positive samples, and RT-PCR analysis typed 26 (84%) of 31 RT-PCR-positive samples. Phylogenetic analysis of the 3' end of the capsid region (363 nucleotides) confirmed the type assignment by EIA and RT-PCR analysis and determined the type for 5 previously untyped samples. Six HAstV antigenic types cocirculated in the community: HAstV-2 (42%), HAstV-4 (23%), HAstV-3 (13%), HAstV-1 (10%), HAstV-5 (6%), and HAstV-7 (6%). RT-PCR and sequence analysis provided more detailed epidemiology of HAstV in the community than did antigenic detection methods.

Molecular characterization of a novel recombinant strain of human astrovirus associated with gastroenteritis in children.

We report a naturally occurring human astrovirus (HAstV) strain detected in two different geographic locations. We identified two isolates of this strain in a diarrhea outbreak at a child care center in Houston, Texas; and two isolates in diarrhea stool samples from two children in Mexico City. All four isolates were detected in stool samples by enzyme immunoassay (EIA). One of the Mexican isolates was typed by EIA and all four isolates were HAstV-5 by typing RT-PCR. The four isolates were >97% nucleotide-identical in two different genomic regions: ORF1a (246 nt), and the 3' end of the genome (471 nt). One isolate from each geographic location was further sequenced in the transition region from ORF1b to ORF2 (1255 nt) and this region of the two isolates showed > or = 99% nt identity. Phylogenetic analyses of sequences of eight HAstV antigenic types and the novel strain in the transition region demonstrated the new strain being closely related to HAstV-3 in ORF1b, but closest to HAstV-5 in ORF2. These results and high sequence identity among all HAstV antigenic types in the transition region and RNA structural predictions supported a potential recombination site at the ORF1b/ORF2 junction. This is the first evidence that recombination occurs among human astroviruses.

Human milk oligosaccharides: a novel method provides insight into human genetics.

Human milk is a unique reservoir of oligosaccharides. The presence of many of these oligosaccharides is determined genetically and is related to the Lewis blood group and secretor antigen status of each donor. A method to quantitate neutral human milk oligosaccharides was developed. Sample preparation was based on a single centrifugation-filtration step that yields oligosaccharide extracts. These extracts first were fractionated to remove a significant portion of their lactose content and were analyzed using high-pH anion-exchange chromatography. Oligosaccharide profiles from 386 milk samples obtained in this fashion generated quantitative information on lactose, the neutral cores lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNneoT), and the key fucosylated oligosaccharides. Additionally, the profiles provided genetic footprints of the Lewis and secretor status of the donors. Furthermore, unusual profiles that could not have been predicted from known genotypes were found. For this reason, milk glycoproteins were studied using carbohydrate-binding probes. Results confirm that oligosaccharides are an accurate predictor of the Lewis blood group status of the donor, and that glycosyltransferases have exquisite specificities. The data obtained in this study corroborate that Lewis-related antigens are tissue specific. This attribute of immunodominant carbohydrate sequences has significant implications for epidemiological studies of breast-fed infants.

Serum antibody as a marker of protection against natural rotavirus infection and disease.

To determine whether naturally acquired serum IgA and IgG antibodies were associated with protection against rotavirus infection and illness, a cohort of 200 Mexican infants was monitored weekly for rotavirus excretion and diarrhea from birth to age 2 years. Serum samples collected during the first week after birth and every 4 months were tested for anti-rotavirus IgA and IgG. Children with an IgA titer >1:800 had a lower risk of rotavirus infection (adjusted relative risk [aRR], 0.21; P<.001) and diarrhea (aRR, 0. 16; P=.01) and were protected completely against moderate-to-severe diarrhea. However, children with an IgG titer >1:6400 were protected against rotavirus infection (aRR, 0.51; P<.001) but not against rotavirus diarrhea. Protective antibody titers were achieved after 2 consecutive symptomatic or asymptomatic rotavirus infections. These findings indicate that serum anti-rotavirus antibody, especially IgA, was a marker of protection against rotavirus infection and moderate-to-severe diarrhea.

Guidelines for the microbiological quality of treated wastewater used in agriculture: recommendations for revising WHO guidelines.

Three different approaches for establishing guidelines for the microbiological quality of treated wastewater that is reused for agriculture are reviewed. These approaches have different objectives as their outcomes: the absence of faecal indicator organisms in the wastewater, the absence of a measurable excess of cases of enteric disease in the exposed population and a model-generated estimated risk below a defined acceptable risk. If the second approach (using empirical epidemiological studies supplemented by microbiological studies of the transmission of pathogens) is used in conjunction with the third approach (using a model-based quantitative risk assessment for selected pathogens) a powerful tool is produced that aids the development of regulations. This combined approach is more cost-effective than the first approach and adequately protects public health. The guideline limit for faecal coliform bacteria in unrestricted irrigation (< or = 1000 faecal coliform bacteria/ 100 ml) is valid, but for restricted irrigation < or = 10(5) faecal coliform bacteria/100 ml is recommended when adult farmworkers are exposed to spray irrigation. A limit of < or = 10(3) faecal coliform bacteria/100 ml is recommended if flood irrigation is used or children are exposed. The guideline limit for nematode eggs for both types of irrigation is adequate except when conditions favour the survival of nematode eggs and where children are exposed; in these cases it should be reduced from < or = 1 egg/l to < or = 0.1 egg/l.

Risk factors for Giardia intestinalis infection in agricultural villages practicing wastewater irrigation in Mexico.

This study assessed the risk factors for Giardia intestinalis infection in an agricultural population in Mexico. Exposure groups included 2,257 individuals from households exposed to untreated wastewater, 2,147 from a group using the effluent from a series of reservoirs, and 2,344 from rain-fed agricultural villages. Stool samples were collected from 6,748 individuals. Wastewater samples were tested for fecal coliforms/100 ml and Giardia sp. cysts/L. Untreated wastewater samples contained 10(8) fecal coliforms/100 ml and up to 300 Giardia sp. cysts/L. Hydraulic retention (3-7 months) in the reservoirs, however, provided an improved effluent quality (10(1)-10(4) fecal coloforms/100 ml and < or = 5 Giardia sp. cysts/L). Children 1-14 years of age had the highest prevalence of infection (20%). Data showed marginal associations between storing drinking water in unprotected containers and lack of facilities for feces disposal and the risk of infection (odds ratios [ORs] = 1.76 and 1.19, 95% confidence intervals [CIs] = 0.95-3.23, and 0.97-1.45, respectively). Individuals purchasing vegetables at the city market had higher rates of infection than those buying at the village shop (OR = 2.49, 95% CI = 1.00-6.17). No excess risk was found in individuals exposed to untreated wastewater compared with controls (OR = 1.07, 95% CI = 0.84-1.36); the group using reservoir water was not different from the controls (OR = 1.22, 95% CI = 0.94-1.58). No risk from agricultural activities was detected (OR = 0.83). This pattern of infection may be addressed by primary health care and wastewater treatment.