Rama Chaudhry

Effects of oral Lactobacillus GG on enteric microflora in low-birth-weight neonates

Background Colonization patterns, especially by anaerobic flora, may play an important role in neonatal gut function. Probiotics could affect disease risk either directly through colonization or indirectly by promoting changes in gut microbial ecology. Methods To study the ability of Lactobacillus GG (LGG) to colonize the neonatal gut and modify its microbial ecology, a prospective, randomized study was performed in 71 preterm infants of less than 2000 g birth weight. Infants less than 1500 g (24 treated, 15 control) received 109 LGG orally twice daily for 21 days. Those infants weighing 1500 to 1999 g (23 treated, 9 control) were treated for 8 days. Stools were collected before treatment and on day 7 to 8 (and day 14 and 21, in the infants weighing less than 1500 g) for quantitative aerobic and anaerobic cultures. Results Colonization with LGG occurred in 5 of 24 (21%) infants who weighed less than 1500 g versus 11 of 23 (47%) in larger infants. Colonization was limited to infants who were not on antibiotics within 7 days of treatment with LGG. There was a paucity of bacterial species at baseline, although larger infants had more bacterial species (1.59 ± 0.13 (SEM) vs 1.11 ± 0.12;P < 0.03) and higher mean log colony forming units (CFU) (8.79 ± 0.43 vs 7.22 ± 0.63;P < 0.05) compared with infants weighing less than 1500 g LGG. Treatment in infants weighing less than 1500 g resulted in a significant increase in species number by day 7, with further increases by day 21. This increase was mainly the result of increased Gram (+) and anaerobic species. No difference in species number was noted in controls. Mean log CFU of Gram (−) bacteria did not change in treated infants weighing less than 1500 g. However, Gram (+) mean log CFU showed a significant increase on day 21 (6.1 ± 0.9) compared with day 0 (3.5 ± 0.9) (P < 0.05). No significant changes in species number or quantitative counts were noted after LGG treatment in the infants weighing 1500 to 1999 g LGG was well tolerated in all infants. Conclusion The neonatal response to a probiotic preparation is dependent on gestational and post-natal age and prior antibiotic exposure. Although LGG is a relatively poor colonizer in infants, especially those infants weighing less than 1500 g at birth, it does appear to affect neonatal intestinal colonization patterns.

A randomized synbiotic trial to prevent sepsis among infants in rural India

Sepsis in early infancy results in one million annual deaths worldwide, most of them in developing countries. No efficient means of prevention is currently available. Here we report on a randomized, double-blind, placebo-controlled trial of an oral synbiotic preparation (Lactobacillus plantarum plus fructooligosaccharide) in rural Indian newborns. We enrolled 4,556 infants that were at least 2,000 g at birth, at least 35 weeks of gestation, and with no signs of sepsis or other morbidity, and monitored them for 60 days. We show a significant reduction in the primary outcome (combination of sepsis and death) in the treatment arm (risk ratio 0.60, 95% confidence interval 0.48–0.74), with few deaths (4 placebo, 6 synbiotic). Significant reductions were also observed for culture-positive and culture-negative sepsis and lower respiratory tract infections. These findings suggest that a large proportion of neonatal sepsis in developing countries could be effectively prevented using a synbiotic containing L. plantarum ATCC-202195.

Long-term colonization of a Lactobacillus plantarum synbiotic preparation in the neonatal gut.

Background: Probiotic, prebiotic, and synbiotic (a combination of pro- and prebiotic) supplements increasingly are being used to prevent and treat a variety of health conditions. Although colonization is considered a key element in the success of such treatments, few clinical studies have addressed colonizing ability. Studies are even more limited in neonates and infants, who may benefit most from such treatment. The present study was conducted to determine the colonizing ability, tolerance, and impact on the stool flora of 7 days of administration of a synbiotic supplement to a neonatal cohort, in preparation for a larger hospital-based trial. Patients and Methods: In this randomized, double-masked, controlled trial, healthy inborn newborns >35 weeks of gestational age and >1800 g birth weight were randomized between 1 and 3 days after birth to receive an oral synbiotic preparation (Lactobacillus plantarum and fructooligosaccharides) or a dextrose saline placebo. Two babies were treated with the synbiotic preparation for every 1 baby treated with the placebo. Duration of therapy was 7 days. Comprehensive stool cultures were done at baseline and on days 3, 7, 14, 21, and 28. Results: Nineteen infants received the active study supplement and 12 infants received the placebo for 7 days. L plantarum was cultured from the stools of 84% of the treated infants after 3 days of treatment, and from 95% of infants on day 28 after birth. Of the infants, 100%, 94%, 88%, 56%, and 32% remained colonized at months 2, 3, 4, 5, and 6, respectively. In both groups, the total mean number of species and the mean log colony counts increased over time. The number of bacterial species was significantly higher on days 21 and 28 in the synbiotic preparation group compared with placebo (P = 0.002 and 0.03, respectively). There was a linear increase in the mean log gram-negative colony counts in the placebo group during the 4-week period that was significantly higher than that in the Lactobacillus group on days 14, 21, and 28 (P < 0.001 for each). In contrast, the supplement group had significantly higher gram-positive colony counts on days 14 (P = 0.002) and 28 (P = 0.04). Only 1 infant in the placebo group was colonized with L fermentum during the first 28 days of life. No difference was found in the percent increase in weight between baseline and day 7, but on day 28 and months 2, 3, and 6, the percent increase from baseline was higher in the probiotic-treated group (P ≤ 0.05). The supplement was tolerated well. Conclusions: The synbiotic preparation colonized quickly after 3 days of administration and the infants stayed colonized for several months after therapy was stopped. There was an increase in bacterial diversity and gram-positive organisms and a reduction of gram-negative bacterial load in the treatment group. Because a combination preparation was used, it is difficult to specifically attribute the colonization to either the probiotic or prebiotic component in this study. Larger efficacy trials are warranted to examine the mechanism of action and precise effects of these supplements.

Fatal Case of Salmonella enterica subsp. arizonae Gastroenteritis in an Infant with Microcephaly

ABSTRACT Salmonella enterica subsp. arizonae is a common gut inhabitant of reptiles, with snakes as the most common reservoir. Though human cases due to this organism are exceedingly rare, it may infect young infants and immunocompromised individuals with a history of intimate associations with reptiles. Gastroenteritis is the most common presentation; others include peritonitis, pleuritis, osteomyelitis, meningitis, and bacteremia. We report a fatal case of S. enterica subsp. arizonae gastroenteritis in a 3-month-old child with microcephaly, with a review of earlier cases and problems encountered in identification of this rare human pathogen.

Emerging Leptospirosis, North India

To the Editor: We read with interest the article, The Changing Epidemiology of Leptospirosis in Israel, published in volume 7, no. 6 (1). Leptospirosis, a septicemic zoonosis with multisystemic involvement, is caused by the pathogenic strains of Leptospira interrogans. Rural farm workers are at high risk for leptospirosis, and it can be a significant public health problem when water and food safety are not ensured. Several epidemics of leptospirosis have occurred on Andaman and Nicobar islands and in southern and western parts of India during the past century (2). The organism has been detected in farm animals in many parts of the country (3); however, human infections have been more or less localized. In 1998, researchers warned that, unless adequate public health measures were initiated, large leptospirosis epidemics were possible in areas where the disease had not been previously reported (4). In addition, they recommended improving clinical diagnosis and conducting systematic epidemiologic studies to control of the disease (4). The true incidence of human leptospirosis in northern India is not known either because of a lack of awareness on the part of the treating physicians or the lack of diagnostic techniques. In 1966, human leptospirosis was reported in Delhi, a state in northern India (5). In a 1966 study (5), sera from persons with pyrexia and jaundice were tested by the agglutination lysis test for leptospiral antibodies. Of 93 serum specimens from persons with pyrexia cases, 3 were positive (1 with L. icterohemorrhagica and two with L. canicola); of 43 serum specimens from persons with jaundice, 3 were positive (2 with L. icterohemorrhagica and 1 with L. icterohemorrhagica and L. pomona). No other study on leptospirosis has been done in the region, and no data are available concerning the problem. To assess the current status of transmission in Delhi and its adjoining areas, we conducted a systematic study for the diagnosis of leptospirosis in our hospital from April 2000 to March 2001; case definition criteria suggested in a previous study (4) were used. A case was defined as a person with fever, headache, and myalgia and more than two of the following symptoms: jaundice, oliguria, respiratory symptoms (cough, hemoptysis, and breathlessness), hemorrhagic manifestations (hematemesis, bleeding gums, and subconjunctival hemorrhage), and signs of meningeal irritation and convulsion. Seventy-five patients (44 male patients; 3–73 years of age) satisfied the inclusion criteria. In addition to clinical evaluation and assessment for other diseases, leptospirosis was investigated by the following laboratory methods: isolation of Leptospira interrogans, direct visualization of the organism under dark-field microscopy, and enzyme-linked immunosorbent assay (ELISA) for Leptospira immunoglobulin (Ig) M antibody (Serion Immunodiagnostica GmbH, Wurzburg, Germany). Per manufacturer’s specifications, the sensitivity, specificity, positive predictive value, and negative predictive value of this kit are 96%, 97%, 90%, and 99%, respectively). All blood samples were sent to the Leptospira referral laboratory at the Indian Veterinary Research Institute, Izzatnagar, for microscopic agglutination test (MAT). Eight serovars of L. interrogans (australis, autumnalis, pomona, sejroe, tarassovi, icterohaemorrhagica, hebdomadis, and patoc) were tested, and a agglutination titer of more than 1:100 was considered positive. All patients were treated empirically with broad-spectrum antibiotics as well as specific drugs according to the results of investigations. Thirty-two patients (42.6%) had a positive ELISA test for Leptospira IgM antibody. The results of MAT were positive in 21 (65.6%) of the 32 ELISA-positive serum samples. Serum specimens from 11 patients reacted with a single serovar, and specimens from 10 patients reacted with more than one serovar. Among the pathogenic species, Leptospira antibodies were detectable by MAT predominantly against L. sejroe (7 of 21), followed by L. icterohaemorrhagica (6 of 21), L. hebdomadis (4 of 21), and L. tarassovi (4 of 21). Leptospira antibodies were also detectable against L. autumnalis (3 of 21), L. australis (2 of 21), and L. pomona (1 of 21). Against L. patoc, MAT could detect antibodies in six samples. The organism could not be isolated in culture or visualized under dark-field microscopy in any of the specimens. Of the 43 case-patients with ELISA-negative specimens, alternative diagnoses were established for 40 on the basis of various laboratory investigations. In five of the case-patients with ELISA-positive specimens, coinfection with other pathogens was detected, including Salmonella typhi (one case) by a positive Widal test, hepatitis C virus by positive ELISA (two cases), and Plasmodium falciparum (two cases) by a positive smear. Five patients, including three who were ELISA positive for Leptospira, died. The highest number of ELISA-positive serum samples (21 of 32) were obtained in August and September 2000, suggesting an epidemic. Epidemiologic investigation of leptospirosis is often hampered by the difficulty of making a definitive microbiologic diagnosis. Isolation of leptospira from clinical samples provides a definitive diagnosis; however, the value of culture is limited because samples have to be collected before the administration of antibiotics, and culturing requires prolonged incubation. Demonstration of typical motility of leptospira under dark-ground illumination in clinical samples, though helpful in early diagnosis, has low sensitivity and depends on the technician’s opinion. Measurement of IgM antibodies against Leptospira by ELISA has emerged as a reliable diagnostic test with good specificity and sensitivity (6). The probability of achieving a positive serologic test increases with the duration of disease, and good correlation between results of MAT and ELISA has been reported by Cumberland et al. (7). MAT has emerged as a dependable diagnostic tool for leptospirosis (next to isolation) by providing serovar specific diagnosis. However, a large number of serovars of L. interrogans exist, and maintaining large numbers of organisms for MAT is difficult for most laboratories. Moreover, MAT may fail to detect antibodies when specific serovars are not used. In this study, the ELISA-positive samples, for which MAT results were negative, may have been caused by infection with serovars other than those used in this study. Because of the problems with methods, leptospirosis is grossly underdiagnosed. Leptospira organisms require humid weather for their survival. Rodents and domestic animals (i.e., cattle and dogs) harbor leptospires and shed the bacteria in urine; they may disseminate the organism in the rain and drinking water sources. Humans frequently come into contact with contaminated water during floods; the number of cases is higher during and after heavy rainfalls. We found that the peak incidence of the disease was during August and September, the monsoon season, which may explain the high incidence of seropositivity during this period. Though the organism has been detected in farm animals in northern India, human leptospirosis has not been considered a major public health problem, probably because transmission is low in arid weather conditions. As a result of 13 consecutive monsoons of above-average strength in India, changes in the environment may be promoting the transmission of this organism. Recently, two other regions in northern India, Chandigarh (8) and Varanasi (9), have reported a Leptospira seroprevalance rate of 8.8% and 21.74%, respectively. Our study supports the warning from other researchers regarding the threat of leptospirosis in areas such as northern India. Preventive measures should be initiated and rapid and definitive diagnostic tests must be developed.

Incidence of Clostridium difficile infection: a prospective study in an Indian hospital

Clostridium difficile is the commonest cause of hospital-acquired diarrhoea. A prospective study comprising of 156 patients and 54 healthy controls was undertaken to assess C. difficile associated diarrhoea (CDAD) incidence in an Indian hospital. Methods used included C. difficile culture and enzyme linked immunosorbent assay (ELISA) for Toxin A. Attempts were made to type isolates by antibiogram and SDS-PAGE. Of the 210 stool samples tested, 12 gave positive results in at least one assay. Of these, 11 were positive by the ELISA method, eight by culture, and seven by both methods. Neither the organisms nor the toxin was found in healthy controls or neonates. The average disease incidence of CDAD estimated by using both methods was 15%. Two antibiotypes of the isolates were obtained and of the isolates characterized by SDS-PAGE, two had identical patterns. This study shows that CDAD is an emerging problem in Indian hospitals. Monitoring should enable the development and implementation of policies and procedures that minimize the risk of this nosocomial pathogen.

Prevalence ofMycoplasma pneumoniae andChlamydia pneumoniae in children with community acquired pneumonia

AbstractA prospective one year study was performed on 62 children admitted at the Alt India institute of Medical Sciences with community acquired pneumonia (CAP) for the prevalence ofMycoplasma pneumoniae andChlamydia pneumoniae. Diagnosis of infection withM. pneumonias was based on serological tests viz microparticle agglutination test for detection of IgM antibodies and indirect immunofluorescence test for antigen detection from throat swabs (sensitivity 85.7%, specificity 93.3%). The indirect solid-phase enzyme immunoassay for detection of IgG antibodies was used to determine the prevalence ofC. pneumoniae (sensitivity 88.8%, specificity 75.8%).Seventeen patients (27.4%) were found to have serotogical evidence ofM. pneumoniae infection whereas only 4 (6.4%) patients were seropositive forC. pneumoniae. Results of this study indicate thatM. Pneumoniae piays a significant role in CAP in infants and young children. Thus specialized laboratory testing for these agents should be more widely used thereby affecting empiric antibiotic regimens.

Adhesion proteins of Mycoplasma pneumoniae.

Mycoplasmas are unique cell wall deficient, cholesterol requiring, highly pleomorphic bacteria that possess very small genome. M. pneumoniae is an important human pathogen that possesses specialized tip organelle to mediate cytadherence. It is a complex multifactorial process requiring a group of mycoplasma proteins such as P1, P30, P116 and HMW1-3. Expression of major mycoplasma adhesin proteins in heterologous expression systems provides an opportunity to study the role(s) of these proteins in pathogenicity and helps to develop diagnostic reagents. In this review we highlight the role(s) of various cytadhesin proteins of M. pneumoniae.

Extended-spectrum β-lactamase-producing Gram-negative bacteria causing neonatal sepsis in India in rural and urban settings

Extended-spectrum β-lactamase (ESBL)-producing Gram-negative bacilli (GNB) are of increasing clinical concern in all age groups worldwide. Whilst sepsis continues to be the leading cause of morbidity and mortality in Indian neonates in the community, identification of microbiological attributes in this population is lacking. This population-based study enrolled 1738 infants with a diagnosis of clinical sepsis at four participating centres in India. Each study site conducted Bactec blood culture, identified bacterial species by API test and stored isolates at -70 °C. From 252 GNB isolates, 155 (113 Klebsiella species, 21 Escherichia coli and 21 other) were subjected to drug susceptibility testing, ESBL phenotyping and testing for clonal relatedness of ESBL strains by PFGE. The results demonstrated that Klebsiella species and E. coli are the most common GNB causes of neonatal sepsis in India, and over one-third are ESBL producers in both community and hospital settings. ESBL-producing strains exhibited frequent co-resistance to aminoglycosides and ciprofloxacin, but remained susceptible to imipenem. PFGE analysis revealed extensive genetic diversity within the ESBL-producing isolates, showing multiple profiles (total of 23). Over 40% of all ESBL-producing isolates formed three pulsed-field profiles (PFP I-III), with PFP-II being the largest cluster (>20% of all ESBL-producing isolates), sharing strains from two distant locations. Identification of a common clone at two geographically distant centres indicated that predominant clones with increased virulence may exist, even in the absence of any clear outbreak. The presence of ESBL-producing strains in community infants with no prior history of hospitalization or antibiotic use dictates heightened vigilance and further studies on the ecology of these organisms.

Clinical and molecular characteristics of nosocomial meticillin-resistant Staphylococcus aureus skin and soft tissue isolates from three Indian hospitals

We analysed risk factors for nosocomial meticillin-resistant Staphylococcus aureus (MRSA) skin and soft tissue infections (SSTIs) in three Indian hospitals. We also determined antimicrobial resistance patterns and genotypic characteristics of MRSA isolates using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and staphylococcal cassette chromosome (SCCmec) typing. Medical records of 709 patients admitted to three tertiary hospitals with nosocomial S. aureus SSTIs were clinically evaluated. Antimicrobial susceptibility testing of patient isolates was performed in accordance with Clinical and Laboratory Standards Institute guidelines, with meticillin and mupirocin resistance confirmed by multiplex polymerase chain reaction. PFGE analysis of 220 MRSA isolates was performed, followed by MLST and SCCmec typing of a selected number of isolates. MRSA was associated with 41%, 31% and 7.5% of infections at the three hospitals, respectively. Multiple logistic regression analysis identified longer duration of hospitalisation [odds ratio (OR): 1.78; OR: 2.83 for >or=20 days], intra-hospital transfer (OR: 1.91), non-infectious skin conditions (3.64), osteomyelitis (2.9), neurological disorders (2.22), aminoglycoside therapy (1.74) and clindamycin therapy (4.73) as independent predictors for MRSA SSTIs. MRSA isolates from all three hospitals were multidrug resistant, with fifteen clones (I-XV) recognised. A majority of the strains possessed type III cassette. The common sequence type (ST) 239 was considered the signature MLST sequence for PFGE clone III. This major MRSA clone III was closely related to the UK EMRSA-1 and was significantly more resistant to antibiotics. Dissemination of multidrug-resistant MRSA clones warrants continuous tracking of resistant genotypes in the Indian subcontinent.