M. Aminul Islam

Phenotypic and Genotypic Characterization of Serologically Atypical Strains of Shigella flexneri Type 4 Isolated in Dhaka, Bangladesh

ABSTRACT Twenty-one atypical Shigella flexneri type 4 strains isolated from patients attending the Dhaka treatment center of the International Centre for Diarrhoeal Disease Research, Bangladesh, were extensively characterized and compared with S. flexneri serotypes 4a and 4b. The atypical strains agglutinated only with the type antigen factor 4 and did not agglutinate with any group factors, thereby excluding their characterization into serotype 4a or 4b. Of the 21 strains, 85.7% did not ferment mannitol but were able to ferment most of the sugars, whereas the remaining 14.3% strains fermented mannitol but were unable to ferment most of the sugars. Most of the strains were resistant to ampicillin, tetracycline, and trimethoprim-sulfomethoxazole. All of the strains harbored the 140-MDa plasmid, had the ipaH gene, had the sen gene (encoding Shigella enterotoxin 2), had the ability to bind Congo red, and were positive for keratoconjunctivitis in the guinea pig eye, attesting their invasive properties. All of the strains contained a middle-range plasmid (35 to 62 MDa) as well as a number of stable small plasmids, yielding mainly two plasmid profiles which were different from those of 4a and 4b strains. Conjugation and curing experiments suggested that the middle-range plasmids harbored a self-transferable multiple antibiotic resistance marker. Pulsed-field gel electrophoresis analysis of all of the tested strains yielded two types with numerous subtypes, whereas ribotyping yielded only two types which were completely different from those of types 4a and 4b. This study concluded that two different clones of atypical S. flexneri type 4 exist and strongly suggests that these are new subserotypes of S. flexneri that await further serological classification.

Phenotypic and genotypic characterization of provisional serotype Shigella flexneri 1c and clonal relationships with 1a and 1b strains isolated in Bangladesh.

ABSTRACT The serotypes of 144 strains of Shigella flexneri serotype 1 (serotypes 1a, 1b, and 1c) isolated from patients attending the Dhaka treatment center of the International Centre for Diarrhoeal Disease Research, Bangladesh, between 1997 and 2001 were serologically confirmed by using commercially available antisera and a panel of monoclonal antibodies specific for S. flexneri group and type factor antigen (MASF). Among serotype 1 isolates, the prevalence of provisional serotype S. flexneri 1c increased from 0 to 56% from 1978 to 2001 in Bangladesh. Detailed biochemical studies revealed that none of the strains of serotype 1 produced indole, while all the strains fermented mannose, mannitol, and trehalose. Twenty percent of the serotype 1c and all the serotype 1a strains fermented maltose and 53% of the serotype 1c strains and 60% of the serotype 1a strains fermented arabinose, whereas all serotype 1b strains were negative for fermentation of these sugars. Only 18% of serotype 1b strains were resistant to nalidixic acid, and most of the serotype 1c and 1b strains were resistant to ampicillin, tetracycline, and trimethoprim-sulfamethoxazole. All the strains of serotypes 1a and 1b and about 88% of the serotype 1c strains were found to be invasive by the Sereny test, had a 140-MDa plasmid, and had Congo red absorption ability. Plasmid profile analysis showed that 26% of the strains of serotype 1 contained identical patterns. Most of the serotype 1c strains (72%) had the 1.6-MDa plasmid, which was not found in either serotype 1a or 1b strains. A self-transmissible middle-range plasmid (35 to 80 MDa) was found in some strains carrying the multiple-antibiotic-resistance gene. Pulsed-field gel electrophoresis analysis yielded three types (types A, B, and C) with numerous subtypes among the serotype 1c strains, whereas serotypes 1b and 1a yielded only one type for each serotype, and those types were related to the types for serotype 1c strains. Ribotyping analysis yielded three patterns for serotype 1c strains and one pattern each for serotype 1a and 1b strains which were similar to the patterns for the serotype 1c strains. Overall analysis of the results concluded that subserotype 1c is closely related to serotypes 1a and 1b. Furthermore, the high rate of prevalence of serotype 1c necessitates the commercial production of antibody against this subserotype to allow the determination of the actual burden of shigellosis caused by provisional serotype 1c.

Death in a Diarrhoeal Cohort of Infants and Young Children Soon After Discharge From Hospital: Risk Factors and Causes by Verbal Autopsy

Assessing mortality pattern of children after discharge from hospital is important to guide appropriate management policy. We studied young children aged 1-23 months, who were discharged from an urban Diarrhoea Treatment Hospital. Children were enrolled on discharge from the hospital, and followed at home after 6 and 12 weeks to assess post-discharge mortality. Of 500 children, 427 were available for evaluation at home 6 weeks after discharge. The median age of the children was eight months, 77 per cent of whom were less than 12 months of age. Of the 427 children, 30 (7 per cent) died within 6 weeks and two died within 12 weeks of discharge from hospital. The median survival time of the deceased was 11 days. Children less than 6 months of age had a five times greater risk of death compared with those aged 6 months or older. Malnutrition, non-breastfeeding, and lack of immunization were important risk factors for death. As ascertained by verbal autopsy, the underlying causes of death were respiratory diseases and watery diarrhoea. Malnutrition and low birth weight were the main associated causes. Hospitalized children, especially young infants, should be given special attention and need to be followed preferably within a week of discharge.

Evaluation of immunomagnetic separation and PCR for the detection of Escherichia coli O157 in animal feces and meats.

Series of animal feces and meat samples artificially contaminated with strains of Escherichia coli O157 isolated from different sources were tested by both an immunomagnetic separation (IMS)-based method and a PCR method using primers specific for a portion of the rfbE gene of E. coli O157. IMS is laborious and time consuming but ends up with the isolation of the pathogen. PCR is fast and less laborious, but it can only be used for screening purposes, so a further culture step is required to isolate the organism. For both fecal and meat samples, the IMS method was found to be more sensitive than the PCR. Furthermore, the detection efficiency of the PCR was influenced by the origin of the fecal sample and the type of meat. For sheep feces, the efficiency of the PCR appeared to be systematically lower than for cattle feces. And the efficiency of the PCR in detecting E. coli O157 in spiked samples of raw minced beef and dry-fermented sausages was systematically lower than in samples of filet americain. Based on this study, it can be concluded that both for animal feces and meat, IMS can be used more successfully to detect E. coli O157 than PCR, because IMS showed to be more sensitive and the outcome was not influenced by the type of animal feces or meat.

Temporal Shifts in the Dominance of Serotypes of Shigella dysenteriae from 1999 to 2002 in Dhaka, Bangladesh

ABSTRACT A total of 358 Shigella dysenteriae strains isolated from patients attending the Dhaka treatment center of the International Centre for Diarrheal Disease Research, Bangladesh, between the years 1999 and 2002 were included in this study. S. dysenteriae type 1, the dominant serotype in 1999 (76.4%), declined to 6.5% in 2002. On the other hand, S. dysenteriae types 2 to 12 were isolated with increasing frequencies of 19, 67, 73.5, and 87% in 1999, 2000, 2001, and 2002, respectively. Of these, types 2 and 4 were the most dominant serotypes, accounting for more than 18.7 and 28.5% of the total isolates, respectively. There was no isolation of serotypes 5, 7, 8, and 13 during this period. Twenty-eight (7.8%) of the isolates were atypical and agglutinated only with the polyvalent antiserum of S. dysenteriae. More than 98% of type 1 strains isolated between 1999 and 2001 were resistant to ampicillin, sulfamethoxazole-trimethoprim, and nalidixic acid. Among other serotypes of S. dysenteriae, Nalr type 2 strains were isolated in 2001 and 2002. Although heterogeneous plasmid profiles were obtained depending on the presence or absence of a single plasmid, core plasmids were defined for particular serotypes. On the other hand, the same plasmid profile was found to be shared by different serotypes. Interestingly, plasmid patterns of types 2 and 4 were almost identical except that a middle-range plasmid of 70 to 60 MDa was present in type 4 in addition to the core plasmids. All the strains harboring the 140-MDa plasmid were positive for the ipaH gene, had Congo red binding abilities, and were positive by the Sereny test, demonstrating their invasive properties.

Zinc status of breastfed and formula-fed infants of different gestational ages

Zinc status in 186 full term and preterm infants was determined at birth, and 3, 6, 9, and 12 months of age along with determination of zinc levels in breast or formula milk to find out if routine zinc supplements are needed during infancy. The leukocyte and plasma zinc levels in all breastfed infants were high at birth and gradually declined reaching lowest values by 4-6 months of age, and improved to normal levels by 9 months following weaning. The preterm infants however, had significantly (P < 0.05) higher leukocyte zinc (213.6 +/- 46.91 micrograms/10(10) cells) at birth compared to term infants. Colostrum of all the mothers had higher zinc concentrations which declined to significantly lower levels in breastmilk by 4-6 months of lactation, corresponding to the age when the breastfed infants had lower zinc levels. The improvement of the levels to normal after weaning suggests that the fall in zinc status during early infancy could be a transient phenomenon which could be reversed by proper weaning, thus strengthening the plea for timely food supplements rather than the need for single nutrient supplements. Formula-fed full term infants had significantly lower leukocyte zinc levels (49.3 +/- 2.59 micrograms/10(10) cells) at 3 months of age compared to breastfed infants of the same age (92.8 +/- 14.04 micrograms/10(10) cells). Even these infants improved their zinc status after weaning on par with breastfed infants. The functional significance of their transient, but low zinc values during early infancy needs to be investigated.

Impact of health education on the feeding of green leafy vegetables at home to children of the urban poor mothers of Bangladesh.

To evaluate the impact of health education on mothers, on the feeding of their children green leafy vegetables (GLV) at home, we studied 160 children aged 6 to 35 months and their mothers in two intervention groups and one comparison group. The mothers of the first intervention group (n = 44) were given health education including a feeding demonstration, by offering a single meal of cooked GLV to their children. The mothers in the second intervention group (n = 36) received health education only. Mothers of both the intervention groups were visited at home after eight weeks of intervention without prior notice, and for each of them an immediate neighbourhood mother having a child in the same age range was selected as a comparison mother (n = 80). During this visit, mothers were asked whether they had cooked GLV that day and fed these to their children; this was confirmed by spot-checking. Also, mothers were interviewed to elicit their perceptions about GLV. The percentages of mothers who thought that GLV are good for health were 88.7%, 86.1% and 76.2% in groups 1, 2 and comparison respectively (P = 0.06). However, the percentages of mothers who actually fed their children GLV were 57%, 64% and 26% in groups 1, 2 and comparison group respectively (P < 0.001). The influence of health education on GLV feeding persisted after controlling for the effect of maternal literacy (Mantel Haenszel chi-square = 16.99; P < 0.0001) and family income (Mantel Haenszel chi-square = 17.36; P < 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)

Leukocyte and plasma zinc in maternal and cord blood : their relationship to period of gestation and birth weight

Zinc is essential for growth. However, its therapeutic use in pregnant women to improve birth weight is not well established. Zinc status of mothers and newborns was determined using paired cord blood and maternal blood samples. Of the newborns investigated, 33 were full term with appropriate weight for date (AFD) (≥2.5 kg) and 30 were either full term (FT) small for date (SFD) (<2.5 kg) or preterm (PT) (<37 weeks gestation) babies. The cord plasma zinc was significantly higher compared with their corresponding maternal levels. However, cord leukocyte zinc was significantly (P<0.01) higher only in PT newborns. Maternal or cord zinc status had no association with birth weight. Plasma copper showed an inverse correlation to plasma zinc. These observations indicate satisfactory zinc status of pregnant women and newborns thus questioning the validity of extra zinc supplements to promote fetal and postnatal growth.

Genotypic and Phenotypic Characterization of Escherichia coli Isolates from Feces, Hands, and Soils in Rural Bangladesh via the Colilert Quanti-Tray System

ABSTRACT The increased awareness of the role of environmental matrices in enteric disease transmission has resulted in the need for rapid, field-based methods for fecal indicator bacteria and pathogen detection. Evidence of the specificity of β-glucuronidase-based assays for detection of Escherichia coli from environmental matrices relevant to enteric pathogen transmission in developing countries, such as hands, soils, and surfaces, is limited. In this study, we quantify the false-positive rate of a β-glucuronidase-based E. coli detection assay (Colilert) for two environmental reservoirs in Bangladeshi households (hands and soils) and three fecal composite sources (cattle, chicken, and humans). We investigate whether or not the isolation source of E. coli influences phenotypic and genotypic characteristics. Phenotypic characteristics include results of biochemical assays provided by the API-20E test; genotypic characteristics include the Clermont phylogroup and the presence of enteric and/or environmental indicator genes sfmH, rfaI, and fucK. Our findings demonstrate no statistically significant difference in the false-positive rate of Colilert for environmental compared to enteric samples. E. coli isolates from all source types are genetically diverse, representing six of the seven phylogroups, and there is no difference in relative frequency of phylogroups between enteric and environmental samples. We conclude that Colilert, and likely other β-glucuronidase-based assays, is appropriate for detection of E. coli on hands and in soils with low false-positive rates. Furthermore, E. coli isolated from hands and soils in Bangladeshi households are diverse and indistinguishable from cattle, chicken, and human fecal isolates, using traditional biochemical assays and phylogrouping.