Subhash Chandra Parija

Ventilator-associated pneumonia: A review

Ventilator-associated pneumonia (VAP) is the most frequent intensive-care-unit (ICU)-acquired infection, with an incidence ranging from 6 to 52% [1,2,3,4]. Several studies have shown that critically ill patients are at high risk for getting such nosocomial infections [3,4]. VAP continues to be a major cause of morbidity, mortality and increased financial burden in ICUs [5,6,7,8]. Over the years there has been a significant advance in our understanding of ventilator associated pneumonia. This article reviews the various aspects of VAP such as definition, risk factors, etiological agents, diagnosis, treatment and prevention with emphasis on the recent advances.

A novel nested multiplex polymerase chain reaction (PCR) assay for differential detection of Entamoeba histolytica, E. moshkovskii and E. dispar DNA in stool samples.

BackgroundE. histolytica, a pathogenic amoeba, is indistinguishable in its cyst and trophozoite stages from those of non-pathogenic E. moshkovskii and E. dispar by light microscopy. We have developed a nested multiplex PCR targeting a 16S-like rRNA gene for differential detection of all the three morphologically similar forms of E. histolytica, E. moshkovskii and E. dispar simultaneously in stool samples.ResultsThe species specific product size for E. histolytica, E. moshkovskii and E. dispar was 439, 553 and 174 bp respectively, which was clearly different for all the three Entamoeba species. The nested multiplex PCR showed a sensitivity of 94% and specificity of 100% for the demonstration of E. histolytica, E. moshkovskii and E. dispar DNA in stool samples. The PCR was positive for E. histolytica, E. moshkovskii and E. dispar in a total of 190 out of 202 stool specimens (94% sensitive) that were positive for E. histolytica/E. dispar/E. moshkovskii by examination of stool by microscopy and/or culture. All the 35 negative control stool samples that were negative for E. histolytica/E. dispar/E. moshkovskii by microscopy and culture were also found negative by the nested multiplex PCR (100% specific). The result from the study shows that only 34.6% of the patient stool samples that were positive for E. histolytica/E. dispar/E. moshkovskii by examination of stool by microscopy and/or culture, were actually positive for pathogenic E. histolytica and the remaining majority of the stool samples were positive for non-pathogenic E. dispar or E. moshkovskii as demonstrated by the use of nested multiplex PCR.ConclusionThe present study reports a new nested multiplex PCR strategy for species specific detection and differentiation of E. histolytica, E. dispar and E. moshkovskii DNA in stool specimens. The test is highly specific, sensitive and also rapid, providing the results within 12 hours of receiving stool specimens.

Role of bone biopsy specimen culture in the management of diabetic foot osteomyelitis.

INTRODUCTION There has been increasing evidence in favor of conservative management of diabetic foot osteomyelitis which requires targeted antibiotic therapy to the causative pathogen. But the method of reliable microbiological isolation is controversial. AIMS AND OBJECTIVES To study the concordance of superficial swab culture with bone biopsy specimen culture in patients with diabetic foot osteomyelitis. MATERIALS AND METHODS A prospective study was conducted from July 2008 to July 2010. All consecutive patients with suspected diabetic foot osteomyelitis were included in the study. Superficial swab and Percutaneous bone biopsy specimens were obtained for culture. The culture results in these two groups were compared for concordance. RESULTS A total of 144 patients were included in the study. 134 cases of bone biopsy specimen and 140 cases of superficial swab showed positive culture results. Mean number of isolate per sample was similar. Staphylococcus aureus was the commonest organism grown in both cultures. The bone pathogen was identified in the corresponding swab culture in only 55 cases (38.2%). Staphylococcus aureus had the highest concordance percentage of 46.5% which was not statistically significant. CONCLUSION Superficial swab culture may not be accurate in identifying all the organisms causing diabetic foot osteomyelitis. Bone biopsy specimen taken simultaneously would increase the accuracy of detecting the bacterial isolate.

EMERGENCE OF VANCOMYCIN INTERMEDIATE STAPHYLOCOCCUS SPECIES IN SOUTHERN INDIA

Since the emergence of meticillin-resistant Staphylococcus aureus (MRSA), vancomycin has been the only uniformly effective treatment for staphylococcal infections in India, teicoplanin and linezolid not being commonly used. The incidence of vancomycin-intermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA) has been increasing in various parts of the world. The first clinical isolate of VRSA was reported from the United States in 2002 (CDC, 2002). More recently, some workers have reported vancomycin-resistant staphylococcal strains from Brazil (Palazzo et al., 2005), Jordan (Bataineh, 2006) and India (Tiwari & Sen, 2006). Vancomycin intermediateresistant Staphylococcus haemolyticus has been reported previously (Schwalbe et al., 1987), and perhaps this is a critical antecedent of VISA. As documented reports of VISA/VRSA in India are very few (Assadullah et al., 2003), we decided to monitor the vancomycin susceptibility of clinical meticillin-resistant Staphylococcus spp. in the tertiary care teaching hospital, JIPMER.

Detection of excretory Entamoeba histolytica DNA in the urine, and detection of E. histolytica DNA and lectin antigen in the liver abscess pus for the diagnosis of amoebic liver abscess

BackgroundAmoebic liver abscess (ALA) and pyogenic liver abscesses (PLA) appear identical by ultrasound and other imaging techniques. Collection of blood or liver abscess pus for diagnosis of liver abscesses is an invasive procedure, and the procedure requires technical expertise and disposable syringes. Collection of urine is a noninvasive procedure. Therefore, there has been much interest shown towards the use of urine as an alternative clinical specimen for the diagnosis of some parasitic infections. Here, we report for the first time the detection of E. histolytica DNA excreted in the urine for diagnosis of the cases of ALA.ResultsE. histolytica DNA was detected in liver abscess pus specimen of 80.4% of ALA patients by a nested multiplex polymerase chain reaction (PCR) targeting 16S-like r RNA gene. The nested PCR detected E. histolytica DNA in all 37 (100%) liver abscess pus specimens collected prior to metronidazole treatment, but were detected in only 53 of 75 (70.6%) pus specimens collected after therapy with metronidazole. Similarly, the PCR detected E. histolytica DNA in 21 of 53 (39.6%) urine specimens of ALA patients. The test detected E. histolytica DNA in only 4 of 23 (17.4%) urine specimens collected prior to metronidazole treatment, but were detected in 17 of 30 (56.7%) urine specimens collected after treatment with metronidazole. The enzyme-linked immunosorbent assay (ELISA) for the detection of lectin E. histolytica antigen in the liver abscess pus showed a sensitivity of 50% and the indirect haemagglutination (IHA) test for detection of amoebic antibodies in the serum showed a sensitivity of 76.8% for the diagnosis of the ALA.ConclusionThe present study for the first time shows that the kidney barrier in ALA patients is permeable to E. histolytica DNA molecule resulting in excretion of E. histolytica DNA in urine which can be detected by PCR. The study also shows that the PCR for detection of E. histolytica DNA in urine of patients with ALA can also be used as a prognostic marker to assess the course of the diseases following therapy by metronidazole. The detection of E. histolytica DNA in urine specimen of ALA patients provides a new approach for the diagnosis of ALA.

Antimalarial drug resistance: An overview.

Malaria is a major public health burden throughout the world. Resistance to the antimalarial drugs has increased the mortality and morbidity rate that is achieved so far through the malaria control program. Monitoring the drug resistance to the available antimalarial drugs helps to implement effective drug policy, through the in vivo efficacy studies, in vitro drug susceptibility tests and detection of molecular markers. It is important to understand the mechanism of the antimalarial drugs, as it is one of the key factors in the emergence and spread of drug resistance. This review summarizes the commonly used antimalarial drugs, their mechanism of action and the genetic markers validated so far for the detection of drug-resistant parasites.

Drug resistance in malaria

Antimalarial chemotherapy is an important component of all malaria control programmes throughout the world. This is especially so in light of the fact that there are no antimalarial vaccines which are available for clinical use at present. Emergence and spread of malaria parasites which are resistant to many of the available antimalarials today is, therefore, a major cause for concern. Till date, resistance to all groups of antimalarials excluding artemisinin has been reported. In recent years, in vitro resistance to even artemisinin has been described. While resistance to antibacterial agents has come to prominence as a clinical problem in recent years, antiparasitic resistance in general and antimalarial resistance in particular has not received much attention, especially in the Indian scenario. The present review deals with commonly used antimalarial drugs and the mechanisms of resistance to them. Various methods of detecting antimalarial resistance and avoiding the same have also been dealt with. Newer parasite targets which can be used in developing newer antimalarial agents and antimalarials obtained from plants have also been mentioned.

Ventilator-associated pneumonia: role of colonizers and value of routine endotracheal aspirate cultures

OBJECTIVES To determine the role of colonizers in the causation of ventilator-associated pneumonia (VAP) and the value of routine pre-VAP endotracheal aspirate (EA) cultures in appropriately treating VAP. METHODS A prospective observational cohort study was conducted over a period of 15 months. Two hundred patients on mechanical ventilation for>48h were studied. RESULTS Acinetobacter spp (33.7%) and Pseudomonas spp (29.8%) were the most common colonizers. Of the 200 patients, 36 developed VAP. In 20 VAP patients, the pre-VAP EA culture-based strategy was not useful. However, in the remaining 16 VAP patients, a pre-VAP EA culture-based strategy would have appropriately treated 13 (81%; 95% confidence interval (CI) 62-100%), in comparison to only nine (56%; 95% CI 32-80%) by the American Thoracic Society (ATS) strategy. The seven patients in whom the ATS guidelines were inappropriate had Acinetobacter spp and Pseudomonas spp resistant to the higher antibiotics recommended by the ATS for multidrug-resistant pathogens. The positive predictive values of Pseudomonas aeruginosa, Acinetobacter baumannii, and methicillin-resistant Staphylococcus aureus (MRSA) isolated from pre-VAP EA cultures were 88%, 83%, and 100%, respectively. CONCLUSION VAP patients should be treated based on ATS guidelines, but whenever P. aeruginosa, A. baumannii, and MRSA are isolated from pre-VAP EA cultures, the initial antibiotic therapy should be extended to treat these.

AmpC beta lactamases among Gram negative clinical isolates from a tertiary hospital, South India

AmpC β-lactamases are cephalosporinases that hydrolyze cephamycins as well as other extended-spectrum cephalosporins and are poorly inhibited by clavulanic acid. Although reported with increasing frequency, the true rate of occurrence of AmpC β-lactamases in different organisms, including members of Enterobacteriaceae, remains unknown. The present study was designed to determine the occurrence of AmpC enzyme-harbouring Gram-negative clinical isolates in a tertiary care hospital in Pondicherry state, South India. A total of 235 Gram negative clinical isolates were tested for resistance to cefoxitin, third generation cephalosporin (3GC) antibiotics, ampicillin, amikacin, co-trimoxazole, gentamicin, meropenem and tetracycline by disc diffusion method. Isolates found resistant to 3GC and cefoxitin were tested for the production of AmpC β -lactamases by three dimensional extraction method and AmpC disc method. Isolates found to sensitive to 3GC were subjected to disc antagonism test for inducible AmpC production. One hundred and thirty four (57%) strains were resistant to 3GC, among which 63(47%) were positive for plasmid-mediated AmpC beta lactamases production. Among the 101 strains sensitive to 3GC, 23 (22.7%) revealed the presence of inducible AmpC beta lactamases by disc approximation test. A total of 80.9% (51/63) of screen positive isolates were detected by Amp C disc test and 93.6% (59/63) by three dimensional extraction method. Out of the 86 AmpC producers, 67 (77.9%) were cefoxitin resistant .Inducible AmpC was not found in Esch.coli and Klebsiella spp. The AmpC producers also concurrently showed multidrug resistance pattern. AmpC producers were found to be prevalent in our hospital and though three dimensional extraction test detects AmpC better, the disk test is easier to perform routinely and is user- friendly.

Evaluation of an IgG-ELISA strategy using Taenia solium metacestode somatic and excretory-secretory antigens for diagnosis of neurocysticercosis revealing biological stage of the larvae.

Diagnosis of neurocysticercosis (NCC) is complicated because of the variability in clinical presentations and course of the disease where viability of parasite is a major determinant. The present study describes evaluation of ELISAs using Taenia solium metacestode somatic and excretory-secretory (ES) antigens for detection of anti-T. solium metacestode IgG antibodies in serum and cerebrospinal fluid (CSF). And results of the ELISAs in cases with a definitive diagnosis of NCC are correlated with the biological stages of the parasite such as live vesicular or degenerated stage. The sensitivity of the IgG-ELISA using ES antigen is observed to be much higher in serum (88.2%) than in CSF (64.28%) although it is only marginally higher in serum (76.4%) than in CSF (75%) when somatic antigen is used in the ELISA. Whereas, the specificities of the ELISA using either somatic or ES antigen for detection of IgG antibodies in serum (97.97%; 96.96%) and CSF (96.42%; 97.61%) are comparable. A strong association is observed between live stage of the parasite and detection of antibodies in sera and CSF from more number of NCC patients by ELISA using ES antigens. Similarly, detection of antibodies by ELISA using somatic antigens could be associated with the dead or degenerated stage of the parasite in brain. The IgG-ELISA strategy developed in the present study opens up an avenue for diagnosis of NCC in hospitals or in population prevalence studies. The use of crude extracts of ES proteins might improve the serodiagnosis of the cases of NCC carrying live vesicular stage of the parasite larvae.