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).

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.

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.

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.

Rapid ethnographic assessment of breastfeeding practices in periurban Mexico City.

Before carrying out a breastfeeding promotion programme in a periurban area of Mexico City, we conducted a rapid ethnographic study to determine the factors associated with absence of exclusive breastfeeding. The responses to pilot interviews were used to develop a standardized questionnaire regarding reasons for infant feeding choice, sources of advice, and barriers to breastfeeding. We interviewed a random sample of 150 mothers with a child < 5 years of age; 136 (91%) of them had initiated breastfeeding; but only 2% exclusively breastfed up to 4 months. The mothers consistently stated that the child's nutrition, health, growth, and hygiene were the main reasons for the type of feeding selected; cost, comfort, and the husband's opinion were less important. Physicians were ranked as the most important source of advice. Reduction or cessation of breastfeeding occurred on the doctor's advice (68%); or when the mothers encountered local folk illnesses such as "coraje" (52%) or "susto" (54%), which are associated with anger or fright; or had "not enough milk" (62%) or "bad milk" (56%); or because of illness of the mother (56%) or child (43%). During childhood illnesses and conditions, breastfeeding was reduced and the use of supplementary foods was increased. This study emphasizes the importance of cultural values in infant feeding choices, defines specific barriers to breastfeeding, and provides a basis for interventions to promote exclusive breastfeeding in the study population.

PIP: Prior to initiating a community-based intervention program to promote exclusive breast feeding in San Pedro Martir, Mexico, a 2-month (1994) rapid ethnographic assessment was conducted. 150 mothers whose youngest child was under 5 years of age were interviewed. 136 mothers (91%) had breast-fed their infant, for a median duration of 6 months, but only 2% exclusively breast-fed for up to 4 months. Mothers consistently described breast feeding as the best nutrition for their infant. However, the dominant feeding pattern was mixed breast and bottle-feeding. Formula, tea, and water were introduced during the first postpartum day. By the end of the third month, 63% of mothers had introduced solid food to promote growth. It was common practice to reduce breast feeding and increase feeding of supplementary foods when a child was ill. Physicians were the most respected source of knowledge on breast feeding. 42% of mothers reported that, at some point when they were breast feeding, a doctor had advised them to stop and half these mothers complied. The data collected in this rapid survey were used to guide a peer counseling program to promote exclusive breast feeding in the community.

A prospective study of astrovirus diarrhea of infancy in Mexico City.

AIM: To describe the epidemiologic and clinical characteristics of astrovirus-associated diarrhea in a cohort of young children from a periurban community in Mexico City. METHODS: From November, 1988, through December, 1991, a total of 214 children were enrolled in a longitudinal study of diarrhea and monitored from birth to 18 months of age. A stool specimen was collected during each episode of diarrhea. Specimens from a total of 510 diarrhea episodes were tested for astrovirus by enzyme immunoassay and examined for other enteric pathogens. The antigenic types of astrovirus were determined by a typing enzyme immunoassay. RESULTS: Astrovirus was detected in 26 (5%) of 510 diarrhea episodes, with an incidence rate of 0.1 episode/child year; the highest rate was in children 13 to 18 months of age. Astrovirus-associated diarrhea was characterized by a median of 4 stools (range, 2 to 10) during the first 24 h, a median duration of 3 days (range, 1 to 21), vomiting (20%), and fever (7%). No cases of dehydration or repeat symptomatic infections were observed. Coinfection with another pathogen was detected in 11 of the 26 episodes (42%). Serotype 2 (35%) was most common, followed by serotypes 4 (15%), 3 (11%), and 1 and 5 (4% each); 31% were nontypable. Astrovirus-associated diarrhea was less severe, as measured by the number of stools (4.3 +/- 1.9), than diarrhea caused by rotavirus (7.1 +/- 2.8) or when coinfections occurred (5.5 +/- 1.6; P = 0.008). CONCLUSIONS: Astrovirus was associated with 5% of the episodes of diarrhea in this cohort of young Mexican children and presented as a mild secretory diarrhea. Five predominant antigenic types were detected with type 2 being the most common.

Role of human-milk lactadherin in protection against symptomatic rotavirus infection.

BACKGROUND: Human milk contains a 46 kDa mucin-associated glycoprotein, lactadherin, which binds specifically to rotavirus and inhibits its replication. This study tested the hypothesis that lactadherin protects against symptoms of rotavirus infection. METHODS: 200 infants in Mexico City were recruited at birth and monitored by regular stool EIA for rotavirus, serology, and recording of feeding and stool patterns. Milk samples were obtained from the mothers weekly until 4 weeks post partum then monthly. The sample taken immediately before an infant's episode of rotavirus infection was assayed for lactadherin, butyrophilin, mucin, and secretory IgA. An infection was defined as symptomatic if diarrhoea occurred in the 5 days before or after detection of the virus. FINDINGS: 31 infants developed rotavirus infection; 15 were symptomatic and 16 had no symptoms. The median concentration of lactadherin in the milk samples (obtained 4-41 days [median 13] before the infection) was 48.4 (range 5.6-180) microg/mL in the asymptomatic group and 29-2 (6.2-103-4) microg/mL in the symptomatic group. Although these medians did not differ significantly, in logistic regression analysis adjusted for age at infection and secretory IgA concentration there was a significant difference between the groups (p=0O01). No association between symptom status and concentrations of butyrophilin, mucin, or secretory IgA was found. INTERPRETATION: Protection against rotavirus by human milk is associated with the glycoprotein lactadherin. This association is independent of products of the secretory immune system.

Milk oligosaccharide profiles by reversed-phase HPLC of their perbenzoylated derivatives.

Human milk is rich in oligosaccharides, some of which inhibit toxins and pathogens involved in diseases of infants. To investigate qualitative and quantitative individual variation of human milk oligosaccharides, a sensitive method for routine identification and quantification of intact milk oligosaccharides was developed and applied to milk samples from 50 donors. The isolated, reduced neutral oligosaccharide fractions were perbenzoylated, resolved by reversed-phase HPLC, and detected at 229 nm. This method resolves most structural isomers and does not require stringent removal of lactose. Peaks were detected at the low nanogram (pmol) level and peak areas were linear from 1 to 1000 micrograms for a standard oligosaccharide. Oligosaccharide samples equivalent to 1 microliter of human milk give optimum chromatographic separation and resolution. The method gives quantitative results comparable to those obtained with classic total sugar analyses, and has an average coefficient of variation of 13%. The 12 major peaks in human milk coeluted with authentic oligosaccharide standards ranging from tri- to octasaccharides, and their identities were confirmed by mass spectrometry. Significant individual variation exists in oligosaccharide profiles; almost 70% of samples contained 2'-fucosyllactose and lacto-N-fucopentaose I as the major oligosaccharides; for the remainder, the major oligosaccharides were 3-fucosytlactose and lacto-N-fucopentaose-II or lacto-N-fucopentaose-III. This method can be used to investigate the extent and biological significance of oligosaccharide variation in human milk.

Effects of thalidomide on HIV-associated wasting syndrome: a randomized, double-blind, placebo-controlled clinical trial.

OBJECTIVE: To evaluate the efficacy of thalidomide in treating wasting syndrome in patients with advanced HIV disease, and to assess the effects of thalidomide on circulating CD4+ T cells, and on HIV viral burden in peripheral blood mononuclear cells (PBMC). DESIGN: Randomized, double-blind placebo-controlled clinical trial. SETTING: Public tertiary care hospital in Mexico City. PATIENTS: Twenty-eight adults with advanced HIV disease being treated with antiretroviral therapy, and who had received antiretrovirals for at least 6 months, who did not have an active opportunistic infection, and who had 10% weight loss in the previous 6 months. INTERVENTIONS: Patients received thalidomide (100 mg by mouth, four times daily) or a matching placebo for the duration of the study (12 weeks). MAIN OUTCOME MEASURES: The main clinical endpoint for efficacy of thalidomide was weight gain or no progression of wasting. Secondary endpoints were Karnofsky performance status, CD4+ cell counts, and HIV viral burden in PBMC. RESULTS: Both groups were comparable in their baseline status. Therapeutic failure occurred in 10 out of 14 patients from the placebo group and in three out of 14 from the thalidomide group (P = 0.021). Weight gain occurred in one patient on placebo and in eight given thalidomide. The Karnofsky index was significantly higher by the end of the study in the thalidomide group (P = 0.003). Mild and transient somnolence and erythematous macular skin lesions were significantly more common in the thalidomide group. CD4+ T cell counts and HIV viral burden in PBMC did not change in either group. CONCLUSIONS: Results suggest that thalidomide not only impeded but also reverted the wasting syndrome, preserving the Karnofsky index in patients with advanced HIV disease. Thalidomide, at the dosage used in this study, had no effect on peripheral CD4+ T cells nor on HIV viral burden in PBMC.