Streptococcal pharyngitis, rheumatic fever and rheumatic heart disease: Eight-year prospective surveillance in Rupnagar district of Punjab, India.

BACKGROUND: Rheumatic fever (RF)/rheumatic heart disease (RHD) continue to be a neglected public health priority. We carried out a registry-based control project, prospective surveillance and sample surveys to estimate the burden of disease. METHODS: We trained healthcare providers and established a surveillance system for the 1.1 million population of Rupnagar district in Punjab. In sample surveys conducted among schools, physicians examined the sampled children. Children with a cardiac murmur were investigated by echocardiography. Throat swabs were obtained from a sub-sample, and group A streptococci (GAS) were identified and emm typed by standard laboratory methods. We estimated the morbidity rates for RF/RHD from surveillance data and school surveys using a correction factor to account for under-registration of cases in the registry. RESULTS: A total of 813 RF/RHD cases were registered from 2002 to 2009. Of the 203 RF and 610 RHD cases, respectively, 51.2% and 36.7% were males. In the age group of 5-14 years, RF was more common (80%) than RHD (27%). The prevalence of RF/RHD in 5-14-year-old students was 1.0/1000 (95% CI 0.8-1.3). The school survey indicated that about two-thirds of the RF/RHD cases were enrolled in the hospital-based registries. Based on the school survey, the prevalence of RF/RHD was estimated to be 143/100,000 population. In the registry, the annual incidence of acute RF was estimated to be at least 8.7/100 000 children in the age group of 5-14 years. The prevalence of GAS was 2% (13/656) in children with sore throat and 0.5% (14/2920) among those not having sore throat. Typing of 27 GAS revealed 16 emm types. We estimate that about 1000 episodes of GAS pharyngitis lead to one episode of acute RF. CONCLUSION: RF/RHD continue to be a public health problem in Punjab, India.

55 kDa outer-membrane protein from short-chain fatty acids exposed Salmonella enterica serovar Typhi induces apoptosis in macrophages.

Various pathogens including Salmonella species are known to induce apoptosis in host cell types during their infection processes. However, the bacterial components capable of inducing apoptosis have not been fully understood. It is now known that in vivo expression of virulence determinants differ from the expression under in vitro conditions. Therefore, in the present study, attempts were made to evaluate the apoptotic potential of outer-membrane protein (OMP) from short-chain fatty acids (SCFA) exposed Salmonella enterica serovar Typhi. Short-chain fatty acids exposure is one of the in vivo stresses encountered by the pathogen in the intestine. Therefore, to simulate the in vivo condition, S. enterica serovar Typhi was grown in the presence of SCFA and its OMP profile was analyzed. The apoptotic potential of 55 kDa protein expressed with enhanced intensity under the SCFA stress was evaluated. Murine peritoneal macrophages interacted with 55 kDa protein showed DNA fragmentation, changes in fluorescence and exposure of phosphatidylserine on their outer leaflets. Levels of nitrite and citrulline were found to be increased in the supernatant of macrophages after interacting them with 55 kDa protein. However, the enzymatic activity of superoxide dismutase was found to be decreased as compared to that of the control (uninteracted) macrophages. These observations indicate that increased levels of nitrite and decreased levels of superoxide dismutase may be one of the mechanisms to induce apoptosis in macrophages by SCFA induced 55 kDa OMP. These findings may help us better understand the pathophysiology of the disease during the host pathogen interactions.

Shiga toxin exposure modulates intestinal brush border membrane functional proteins in rabbit ileum.

The activities of lactase, sucrase and alkaline phosphatase (AP) were studied in intestinal brush border membranes of control and toxin-treated rabbits. Purified Shiga toxin (Stx) exposure to ileal mucosa inhibited activities of brush border enzymes by 50%. Kinetic analysis revealed that the observed decrease in BBM enzyme activities was due to reduced V(max) with no change in the affinity constants of the systems. The observed changes in enzyme activities were corroborated by Western Blot analysis of lactase, sucrase and AP. The mRNA levels encoding sucrase and lactase proteins in control and Shiga toxin-treated rabbit ileum did not show any change in the rabbit ileum. Histopathological analysis showed short, blunt villi with increased number of inflammatory cells in the lamina propria and extrusion of cells in to the lumen of Stx-treated rabbit ileum. The present findings suggest that Shiga toxin act by inhibiting protein synthesis of these brush border functional proteins beyond their transcriptional level and by the direct damage to intestinal epithelium, which could be implicated in the pathogenesis of diarrhea.

Macrophage cell death due to Salmonella enterica serovar Typhi and its acid stress protein has features of apoptosis.

Salmonella spp. have been shown to cause apoptosis of various host cell types as a part of their infection process. However, the induction of apoptosis remains to be looked into under the different host environments including acidic stress experienced by the pathogen. In order to simulate the in vivo acidic conditions, we studied the potential of S. typhi and its protein expressed under in vitro acidic conditions to induce apoptosis in macrophages. Murine macrophages were isolated and interacted with serovar Typhi and its acid stress protein for different time periods. The assessment of nucleosomal DNA, and nuclear staining with H-33342 dye and flow cytometry indicated the occurrence of characteristic features of apoptosis. Analysis of data revealed that S. typhi caused apoptotic cell death in 61% of macrophages whereas stress-induced protein alone accounted for apoptotic cell death in 45% of macrophages. The present study, for the first time demonstrates the potential of stress-induced outermembrane component of S. typhi to induce apoptosis. Identification of such factors may offer new insights for understanding the pathophysiology of the disease during the host-pathogen interactions.

Reactivity of typhoid patients sera with stress induced 55 kDa phenotype in Salmonella enterica serovar Typhi.

Salmonella faces a variety of stresses including acid and heat, in the natural environment whether in the gastrointestinal tract of mammalian host or in the external environment during transmission where survival and multiplication is a priority for the pathogen. In the present study, Salmonella enterica serovar Typhi was grown under acid (inorganic and organic) as well as heat stress and the outermembrane protein (OMP) profiles were compared. A 55 kDa OMP was found to be expressed with high intensity under the selected stress conditions in comparison to normal conditions. The protein expressed under acidic stress reacted with antibodies raised against heat shock protein indicating the similarity of atleast some of the epitopes. In vivo immunogenicity (reactivity with typhoid patient sera) revealed that the 55kDa protein under each stress condition was reactive with 83% of the typhoid sera. In the light of role of the stress induced proteins in pathogenesis of microbial infections and their immunogenic potential, these findings may be relevant for a better understanding of the host-microbe interactions and for future development of diagnostic and preventive strategies.

Evaluation of polymerase chain reaction (PCR) for diagnosing Haemophilus influenzae b meningitis.

A polymerase chain reaction (PCR) for detecting Hib in cerebrospinal fluid (CSF) was evaluated and compared with culture and a latex agglutination test (LAT) in a hospital-based prospective surveillance. We studied 107 children aged from 1 month to 12 years with a clinical and CSF profile suggestive of acute bacterial meningitis. CSF culture was performed on blood-chocolate agar by standard technique, LAT by a commercially available kit (Wellcogen) and PCR using total DNA extracted from CSF samples. Of 107 children, 79% had received one or more doses of injectable antibiotics. Hib was detected by culture in 14 cases, by LAT in 23 and by PCR in 37. All CSF samples that reveal Hib by culture or LAT had a PCR positive for Hib (sensitivity 100%). PCR also detected 14 additional cases of Hib meningitis which were not detected by culture or LAT. We conclude that PCR is a sensitive and specific diagnostic tool that may be valuable in a population with high pre-hospital antibiotic usage.