Benu Dhawan

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.

In vitro antimicrobial activity of alpha-melanocyte stimulating hormone against major human pathogen Staphylococcus aureus

Alpha-melanocyte stimulating hormone (alpha-MSH) is an endogenous anti-inflammatory peptide reported to possess antimicrobial properties, however their role as antibacterial peptides is yet to be established. In the present study, we examined in vitro antibacterial activity of alpha-MSH against S. aureus strain ISP479C and several methicillin-sensitive (MSSA) and methicillin-resistant (MRSA) S. aureus strains. Antibacterial activity was examined by varying several parameters, viz., bacterial cell densities, growth phase, pH, salt concentration, and temperature. Antibacterial activity was also examined in complex biomatrices of rat whole blood, plasma and serum as well as in biofilm form of bacteria. Our results showed that alpha-MSH possessed significant and rapid antibacterial activity against all the studied strains including MRSA (84% strains were killed on exposure to 12 microM of alpha-MSH for 2h). pH change from 7.4 to 4 increased alpha-MSH staphylocidal activity against ISP479C by 21%. Antibacterial activity of alpha-MSH was dependent on bacterial cell density and independent of growth phase. Moreover, antimicrobial activity was retained when alpha-MSH was placed into whole blood, plasma, and serum. Most importantly, alpha-MSH exhibited antibacterial activity against staphylococcal biofilms. Multiple membrane permeabilization assays suggested that membrane damage was, at least in part, a major mechanism of staphylocidal activity of alpha-MSH. Collectively the above findings suggest that alpha-MSH could be a promising candidate of a novel class of antimicrobial agents.

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.

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.

A study of bacteremia in febrile neutropenic patients at a tertiary-care hospital with special reference to anaerobes.

Patients with hematological malignancies who are receiving chemotherapy suffer prolonged periods of neutropenia, which leads to a greater risk of infection and mortality. A prospective study was conduced to determine the incidence of bacteremia in patients of hematological malignancies over a 2-yr period. A total of 119 episodes of febrile neutropenia occurred among 96 consecutive patients, of which 35 episodes were associated with bacteremia. Forty-four percent of the isolated bacteria were Gram-positive aerobes and 46% were Gram-negative aerobes. Staphylococcus aureus, Enterococcus spp., and Escherichia coli were the most common isolates. Gram-negative bacteremia was associated with a higher mortality. Anaerobes accounted for 4.4% of all isolates. The episodes of anaerobic bacteremia were polymicrobial and had a fatal outcome. A high incidence of antimicrobial resistance among aerobic and anaerobic bacteria was also recorded. Compared to previous years, a shift from a predominating Gram-negative to a Gram-positive etiology was noted. The initial empiric antibiotic regimens should be based on a local knowledge of the most common causative microorganisms, their sensitivity pattern, and the outcome of bacteremia.

Association of Campylobacter jejuni infection with childhood Guillain-Barré syndrome: a case-control study.

A prospective case-control study was conducted to determine the association between Campylobacter jejuni infection and childhood Guillain-Barré syndrome in the Indian population. We found evidence of recent Campylobacter jejuni infection in 27.7% of patients with Guillain-Barré syndrome, as compared with 2.3% in neurological controls (P = .003) and 2.3% in nonneurological controls (P = .003). Of the 15 patients with Campylobacter jejuni infection, 8 (53.3%) reported having had diarrhea within 12 weeks before the onset of the neurologic illness. Our results suggest association between recent Campylobacter jejuni infection and bulbar weakness (P = .043). No statistical difference was observed between the Campylobacter jejuni positive and negative groups with respect to age, other clinical features, albuminocytological dissociation, and residual paralysis at follow-up. It is concluded that subclinical Campylobacter jejuni infection is an important antecedent illness in childhood Guillain-Barré syndrome in the Indian population.

Outcome in childhood Guillain-Barré syndrome

ObjectiveTo prospectively assess the outcome of children diagnosed with Guillain-Barré syndrome (GBS), followed up for a median duration of 25 months.MethodsTertiary center, prospective follow up of children with GBS enrolled between, Dec 2003 to Sep 2006. Functional recovery was determined at 12 months and later using Hughes scale (0–6). Clinical, electrophysiological variables were compared between the good outcome (grade 0/1) and bad outcome groups (died or functional grade >1).ResultsAmong 52 children with a median age of five yr there was male preponderance (75.4%). Mortality during acute phase was 11.5% (6/52). Among the survivors long term data was obtainable in 40 of the 46 children. At one year follow up 87.5% children had fully recovered or had minimal symptoms, beyond one year this rose to 95%. Only 2 among 40 had significant symptoms at last follow up (1 grade-2 and 1 grade-3). Factors significantly associated with poor outcome were: need for artificial ventilation, inexitable nerves on nerve conduction testing and delayed independent walking.ConclusionChildren needing ventilation have the worst short-term prognosis. However, irrespective of severity during acute phase, good long-term recovery can be expected in most children.

Association of Mycoplasma pneumoniae and asthma among Indian children

Abstract The role of Mycoplasma pneumoniae infection as a trigger for asthma exacerbations is well supported in previous studies. This study was designed to investigate the role of M. pneumoniae infection in acute exacerbation of asthma in children. A total of 150 patients (110 males, 40 females) were studied and immunoglobulin M (IgM) antibodies to M. pneumoniae were detected by enzyme‐linked immunosorbent assay (ELISA), and PCR amplification was performed for the P1 gene to associate M. pneumoniae infection with asthma. As compared with 33 children with asthma, only two of the control subjects had positive IgM titers for M. pneumoniae, which was statistically significant (P=0.002). A total of 15 children with asthma were positive by PCR for the P1 gene while none of the controls had a positive PCR. Of these positive cases, 24 cases were positive only by ELISA, six were positive only by PCR and nine patients were found to be positive by both ELISA and PCR. All the clinical characteristics of the patients at baseline were comparable between the moderate and the severe group of patients statistically, except for the peak expiratory flow rate. Mycoplasma pneumoniae infection was found to have a significant association with acute exacerbation in the moderate group of asthma patients by PCR (P=0.01). These data suggest that M. pneumoniae infection may contribute to asthma exacerbation.

Childhood Acute Transverse Myelitis: Clinical Profile, Outcome, and Association With Antiganglioside Antibodies

Prospectively, in 15 children the association of acute transverse myelitis with Campylobacter jejuni infection and antiganglioside antibodies was studied. The clinical profile, radiological findings, and treatment outcome in these children were analyzed. Stool culture and serology for Campylobacter jejuni and antiganglioside antibodies were tested. In all, 15 age- and sex-matched healthy controls were tested for evidence of Campylobacter jejuni infection and antiganglioside antibodies. Anti-GM1 antiganglioside immunoglobulin G antibodies were found in 46% of patients with acute transverse myelitis versus 6.6% of controls (P = .035). Evidence of Campylobacter jejuni infection was absent in both the groups. The magnetic resonance imaging revealed longitudinally extensive lesions in majority. All children underwent intravenous high-dose corticosteroid treatment. At 1-year follow-up, 8 children had recovered completely, whereas 3 were nonambulatory. Bladder disturbances persisted in 7. The significance of these findings and the possible role antiganglioside antibodies may play in acute transverse myelitis pathophysiology is discussed.