Comparison of a modified adherence assay with existing assay methods for identification of enteroaggregative Escherichia coli.

Localized, diffuse, and aggregative adherence patterns of Escherichia coli identified with specific DNA probes were compared in cell culture adherence assays by using the Center for Vaccine Development, Baltimore, method, the University of Texas Medical School, Houston (UTH), method, and a modified UTH method. Increasing postwash incubation time from 2 to 4 h in the modified UTH method allowed identification of enteroaggregative E. coli, which was otherwise not identified by the original UTH method.     

Characterization of Aeromonas caviae antigens which cross-react with Shigella boydii 5.

Live and boiled cells of 16 strains of Aeromonas caviae, isolated from patients with diarrhea, agglutinated with Shigella boydii 5 antiserum in a slide test. Further studies with seven selected strains showed agglutination with boiled cells in a tube test. Lipopolysaccharide antigen extracted from one of these strains cross-reacted with S. boydii 5 in enzyme-linked immunosorbent assay and immunoblot studies. Either all or the majority of the seven strains possessed properties deemed to be diarrheagenic.     

Characterization of Aeromonas spp. isolated from humans with diarrhea, from healthy controls, and from surface water in Bangladesh.

Aeromonas isolates from patients with diarrhea in Bangladesh (n = 69), from healthy controls (n = 11), and from surface water (n = 40) were analyzed with respect to their hybridization groups (HGs) by the aid of fatty acid methyl ester (FAME) characterization and DNA fingerprinting by AFLP, biochemical phenotypes (Phe-nePlate [PhP] types), and the production of hemolysin and cytotoxin. The aim of the investigation was to find out whether certain strains carrying virulence factors predominated among patient isolates. According to FAME and/or AFLP analysis, most human isolates were allocated to DNA HGs 4 (Aeromonas caviae) and 1 (A. hydrophila). Most environmental strains were allocated to HG8 (A. veronii biogroup sobria) and HG4 (A. caviae), and only one was of HG1. According to PhP typing, the diversity among patient isolates was lower than that among other strains, and two dominating PhP types (types BD-1 and BD-2) were identified in 29 and 30% of the patient isolates, respectively. PhP type BD-1 was also common among the environmental isolates, whereas PhP type BD-2 was only identified in two of the other isolates. Twenty-five of 26 isolates belonging to HG1 were of the same PhP type (BD-2), whereas isolates of other common HGs were more diverse according to their PhP types. Hemolytic and cytotoxin-producing strains occurred more frequently among the environmental isolates than among patient isolates. However, the hemolytic and cytotoxic activities among human isolates was strongly correlated to the HG1/BD-2 type, which, in addition, showed high cytotoxin titers (median values, 1/512 compared to 1/128 for cytotoxin-positive isolates belonging to other types). Thus, the HG1/BD-2 type may represent a pathogenic A. hydrophila type that is able to produce diarrhea in humans.     

Controlled study of Escherichia coli diarrheal infections in Bangladeshi children.

Diarrheal diseases are highly prevalent in Bangladesh. However, the relative contribution of diarrheagenic Escherichia coli organisms--those that are enterotoxigenic (ETEC), enteropathogenic (EPEC), enteroinvasive, enterohemorrhagic, enteroaggregative, and diffuse adherent--to diarrhea in Bangladeshi populations is not known. With DNA probes specific for these diarrheagenic E. coli strains, we analyzed fecal E. coli from 451 children up to 5 years of age with acute diarrhea seeking treatment at a Dhaka hospital and from 602 matched control children without diarrhea from July 1991 to May 1992. Enteroinvasive E. coli was not isolated from any children; enterohemorrhagic E. coli was not isolated from any diarrheal children but was isolated from five control children; enteroaggregative and diffuse adherent E. coli strains were isolated with similar frequencies from children with and without diarrhea, thereby showing no association with diarrhea; ETEC was significantly associated with diarrhea in the diarrheal children as a whole and especially in the age groups of 0 to 24 months and 37 to 48 months (further analysis suggests an association with diarrhea for the heat-stable toxin only and for both heat-labile- and heat-stable-toxin-producing ETEC only); and EPEC was significantly associated with diarrhea in the diarrhea group as a whole and particularly in infants up to 1 year of age. Further analysis suggested that EPEC strains of only the traditional serogroups were significantly associated with diarrhea. ETEC and EPEC infections peaked during warm months. Our data thus suggest that EPEC and ETEC are important causes of acute diarrhea in children in this setting.     

Presence of hepatitis B surface antigen mutant G145R DNA in the peripheral blood leukocytes of the family members of an asymptomatic carrier and evidence of its horizontal transmission

An asymptomatic carrier and all six of his family members were detected positive for HBV DNA in their peripheral blood leukocytes (PBL), by polymerase chain reaction. Direct sequencing of the amplified DNA revealed that the HBV DNA from the carrier and his wife was of subtype ayw. Interestingly, the amplified HBV DNA from the five other members of the family was found to be not only of subtype adw but also contained G to A mutation at nucleotide position 587. This indicates the presence of established vaccine escape mutant of the virus (G145R) and suggests two different sources of infection within the family. Southern blot hybridization of EcoR1 digested DNA from PBL indicated presence of HBV DNA, integrated into cellular DNA and also in the form of free viral DNA. The study not only establishes the persistence of surface mutant G145R HBV DNA, within the PBL of HBsAg negative individuals from the non-vaccinated random population, but also suggests possible horizontal transmission of the mutant among the family members although none of the family members has received immunoprophylaxis against HBV or had clinically apparent disease or any other known risk factors of HBV infection. As all of them were seronegative for HBsAg/antiHBc, the presence of G145R mutant in the PBL signaled possibility of spread of the vaccine escape mutant virus by blood transfusion, unsafe injection practices or through sexual root.     

Leishmania promastigote membrane antigen-based enzyme-linked immunosorbent assay and immunoblotting for differential diagnosis of Indian post-kala-azar dermal leishmaniasis

Diagnosis of post-kala-azar dermal leishmaniasis (PKDL), caused by Leishmania donovani, is difficult, as the dermal lesions are of several types and resemble those caused by other skin diseases, especially leprosy. Since the disease generally appears very late after the clinical cure of kala-azar in India, it is also difficult to correlate PKDL with a previous exposure to L. donovani. Very few attempts have been made so far to diagnose PKDL serologically, and the diagnostic methods vary in their sensitivities and specificities. Diagnosis of PKDL through sophisticated PCR methods, although highly sensitive, has limited practical use. We have developed a serodiagnostic method using an enzyme-linked immunosorbent assay to detect specific immunoglobulin (Ig) isotypes and IgG subclass antibodies in the sera of Indian PKDL patients. Our assay, which uses L. donovani promastigote membrane antigens, was 100% sensitive for the detection of IgG and 96.7% specific for the detection of IgG and IgG1. Optical density values for individual patients, however, demonstrated wide variations. Western blot analysis based on IgG reactivity could differentiate patients with PKDL from control subjects, which included patients with leprosy, patients from areas where kala-azar is endemic, and healthy subjects, by the detection of polypeptides of 67, 72, and 120 kDa. The recognition patterns of the majority of serum samples from patients with PKDL were also distinct from those of the serum samples from patients with visceral leishmaniasis (VL), at least for a 31-kDa polypeptide. To further differentiate patients with PKDL from those with active and cured VL, we analyzed the specific titers of the Ig isotypes and IgG subclasses. High levels of IgG, IgG1, IgG2, and IgG3 antibodies significantly differentiated patients with PKDL from patients cured of VL. The absence of antileishmanial IgE and IgG4 in patients with PKDL differentiated these patients from those with active VL. These results imply intrinsic differences in the antibodies generated in the sera from patients with PKDL and VL.     

Setting up a Quality Assurance model for newborn care to strengthen health system in Bihar,India

Background

A Quality Assurance model was rolled out in Bihar, India. It had two components: external and internal monitoring and giving feedback for action. The parameters included infrastructure and policy, equipment maintenance, stock supply and aseptic measures.

Methods

The performance and gradation into good/average/poor was measured based on the scores translated from the data collected after giving appropriate weights.

Result

12%, 63%, and 25% units were categorized as good, average and poor based on infrastructure. For equipment, 68% of units performed poorly; for stock maintenance 64% and 35% of NBCCs fell under good and average categories respectively; most (54%) NBCCs had average scores for aseptic measures; 30% fell in the poor category.

Conclusions

Involvement of government in monitoring and feedback mechanism, establishing a system of data collection at the grass root level and analysis at the state level were the positive outcomes.