Molecular Characterization of New Delhi Metallo-ß-Lactamases-Producing Bacteria in the Gastrointestinal Tract of Intensive Care Unit Patients.

BACKGROUND: The emergence of carbapenem-resistance in the gut flora of patients in the intensive care unit (ICU) poses a significant risk for infection with these types of pathogens. MATERIALS AND METHODS: New Delhi metallo-ß-lactamase 1 (NDM-1) in the gut flora of ICU patients was detected in cultures of a single rectal swab from each patient admitted to the ICU for a minimum period of 48 hrs. Samples were processed in the microbiology laboratory using blood agar and MacConkey agar. Identification of pathogens, carbapenem resistance, and metallo-ß-lactamase production was made using standard laboratory procedures. Bacterial isolates were also used for the determination of the NDM-1 gene by molecular methods. RESULTS: One hundred twenty-two patients with different clinical presentations were recruited in the study. Two hundred nine bacteria were isolated, with Escherichia coli being the most common isolate. A total of 54/122 (44.3%) patients harbored carbapenem-resistant organisms (CRO), 36/122 (29.5%) carried metallo-ß-lactamase-producing organisms (MBLO), and 30/122 (24.6%) carried bacteria with the NDM-1 gene. Patients who harbored CRO and MBLO had longer mean duration of stay in the ICU and hospital than those not harboring CRO and MBLO. All the metallo-ß-lactamases were simultaneously resistant to other groups of antibiotics also. Use of invasive devices, three or more classes of antibiotics, hospitalization during the previous six months, comorbidities, and hospital stay for ≥48 hours before ICU admission had a significant association with colonization with CRO. CONCLUSION: Patients admitted in ICU or with serious diseases should be screened for gastrointestinal carriage of carbapenem-resistant organisms. Irrational use of antibiotics must be stopped to prevent the emergence and spread of such organisms.

A Comparative Effectiveness Research of Azathioprine and Cyclophosphamide on the Clinical and Serological Response in Pemphigus Vulgaris.

CONTEXT: A prospective study was carried out to examine the efficacy of cyclophosphamide and azathioprine in pemphigus vulgaris. AIMS: To compare the clinical and serological effect of azathioprine and cyclophosphamide in pemphigus patients. MATERIALS AND METHODS: Prospective, institutional based study was conducted twenty-one patients of pemphigus vulgaris were initiated on either azathioprine (n = 9) or cyclophosphamide (n = 7) in addition to prednisolone and were evaluated clinically (mucosal and cutaneous severity) and serologically enzyme-linked immunosorbent assay (ELISA) at 0, 3 and 6 months. RESULTS: Azathioprine had a slower onset of action with a statistically significant improvement seen by 6 months (P = 0.016). Cyclophosphamide had a faster onset of action (3 months) though there was no statistical difference in the efficacy between the two at the end of 6 months. The (RonT) was 33.3-44.4% for azathioprine and 28.8-42.9% for cyclophosphamide at 6 months. Though ELISA had a high sensitivity and specificity for diagnosis, as a tool for assessing therapeutic response a significant decrease was seen only till 3 months. This was restricted to Dsg1 for the azathioprine group and both Dsg3 and Dsg1 levels for the cyclophosphamide group. There were two deaths, both in the cyclophosphamide group. CONCLUSIONS: Azathiorpine and cyclophosphamide are equally effective for mucosal and cutaneous disease in pemphigus after 6 months of therapy. Dsg ELISA is useful for diagnosis of pemphigus but is not a useful tool for monitoring response to therapy.

Multi drug resistance and Extended Spectrum Beta Lactamases in clinical isolates of Shigella: A study from New Delhi, India.

BACKGROUND: Shigella is an important cause of gastroenteritis in local Indian population, as well as of traveler's diarrhea in the international visitors to India. These patients often require appropriate antimicrobial therapy; however, rapid development of antimicrobial resistance poses a major hurdle in achieving this goal. METHOD: A prospective study was conducted during 2009-12 in New Delhi, India, including 6339 stool samples from gastroenteritis patients. 121 Shigella strains were identified on the basis of colony morphology, biochemical reactions, serotyping and ipaH gene based PCR. Antimicrobial susceptibility testing by disc diffusion, MIC determination by Vitek(®) 2 and phenotypic tests for ESBL/AmpC production were done. RESULTS: Nineteen percent strains (23/121) were found to be resistant to third generation cephalosporins and all were phenotypically confirmed to be ESBL producers; one strain was positive for AmpC. ESBL producing strains were also found to be significantly more resistant (p < 0.05) to several other antimicrobials agents in comparison to ESBL non-producers, [ampicillin (100% vs. 62.2%), ampicillin/sulbactam (100% vs. 30.6%), cotrimoxazole (100% vs. 77.6%), ciprofloxacin (87.0% vs. 49.0%), ofloxacin (87.0% vs. 52.0%) and gentamicin (30.4% vs. 7.1%)]. Multidrug resistance was seen in 76% strains. CONCLUSIONS: Inappropriate use of antimicrobial agents puts high selection pressure on the higher-end antibiotics. Multi-drug resistance and high rates of ESBL production by Shigella is a matter of concern for the local population as well as international travelers. Therefore, better national level antimicrobial management programs are the priority needs.

True Prevalence of Shigellosis in Indian Children with Acute Gastroenteritis: Have We Been Missing the Diagnosis?

BACKGROUND: Shigella is responsible for high morbidity and mortality among children, yet its true prevalence remains inconclusive. The aim of this study was to determine the actual prevalence of Shigella infection in childhood diarrhea and dysentery cases and assess the applicability of ipaH gene PCR in Indian settings. METHODS: This study was conducted at Maulana Azad Medical College and associated Lok Nayak Hospital, New Delhi, India during 2011-12. A total of 385 children (207 with diarrhea, 118 with dysentery, and 60 matched controls) were enrolled. Stool samples were cultured, and the suspected colonies were analyzed using biochemical reactions and serotyping. Antimicrobial susceptibility testing was done using disc diffusion method. ipaH-gene PCR was performed directly on stool samples collected from 180 randomly selected patients (60 from each group). RESULTS: Shigella was isolated using conventional culture methods in 8.2% (95% CI: 5.1%, 12.8%), 33.1% (95% CI: 25.2%, 42.0%), and 0% in the diarrhea, dysentery and control cases, respectively. High resistance was seen towards co-trimoxazole, nalidixic acid, fluoroquinolones, doxycycline and several beta-lactams drugs. Actual prevalence of shigellosis was determined using ipaH gene PCR to be 18.3% (95% CI: 10.4% - 30.1%) diarrhea cases and 56.7% (95% CI: 44.1, 68.4%) dysentery cases. One (1.7%, 95% CI: 0.01%, 9.7%) control specimen also yielded positive result in PCR. CONCLUSIONS: Correct diagnosis of shigellosis is essential to start antimicrobial therapy in selected cases. The prevalence of Shigella / EIEC infection in children is much higher than previously estimated. Despite its high costs and other limitations, we recommend the use of ipaH-gene PCR as a routine tool in the management of childhood acute gastroenteritis cases.

Elevated levels of vascular endothelial growth factor in adults with severe dengue infection.

The immune pathogenesis of dengue involves antibody production, B cell and T cell response and various pro-inflammatory and anti-inflammatory cytokines. VEGF, a potent permeability enhancing cytokine, is thought to play a pivotal role in mediating plasma leakage in DHF. It is a member of growing family of related proteins that includes VEGF B, VEGF C, VEGF D and placental growth factor. It promotes angiogenesis and vascular integrity. In addition to its role in promoting endothelial permeability & proliferation, it may contribute to inflammation and coagulation. This study was undertaken to investigate the role of VEGF in the patients with dengue infection. Sera were collected from 106 patients with various grades of dengue illness and 40 healthy controls and tested for VEGF levels using commercial ELISA kits. Viral serotypes were detected using specific primers. The results showed very low levels of VEGF (3.493 ± 1.982 pg/ml) in healthy controls. Levels of VEGF were higher in patients with severe dengue (428.170 ± 224.61 pg/ml) as compared to patients with non severe dengue with and without warning signs (290.407 ± 167.17 pg/ml). Significant correlation (p < 0.001) was found between raised VEGF levels and thrombocytopenia and raised haematocrit levels. The VEGF profile patterns discovered between the different phases of illness indicate an essential role in dengue pathogenesis and with further studies may serve as predictive markers for progression of dengue fever to severe dengue infection.

A comparative study of bacterial and parasitic intestinal infections in India.

BACKGROUND: Infectious diarrhea causes a major health problem in developing countries with significant morbidity and mortality. Very often, rehydration therapy alone does not suffice, mandating the use of antimicrobial agents. However, rapidly decreasing antimicrobial susceptibility is complicating the matters. MATERIALS AND METHODS: The study aimed to determine the prevalent bacterial and parasitic agents of diarrhea in India. A cross-sectional study was done at Maulana Azad Medical College and associated Lok Nayak Hospital, New Delhi, during 2012-14. Stool samples were received from patients of all age groups and processed for bacteriological and parasitological identification by microscopy, bacterial culture, biochemical identification, serotyping and antimicrobial susceptibility tests. The study also aimed to identify the recent papers (after year 2000) reporting aetiology of infectious diarrhea in India involving the general population as a whole and compare them with present findings. RESULTS: Out of 6527 samples, 581 (8.90%) were positive for bacterial pathogens. A total of 280 samples (of 3823 under-five year children) were positive for diarrheagenic Escherichia coli. Other organisms like Vibrio cholera were found in 159 (2.44%) cases, Shigella spp. in 126 (1.93%), Salmonella Typhi in 7 (0.11%), Salmonella Typhimurium in 6 (0.10%), Aeromonas hydrophila in 3 (0.05%) cases. Levels of resistance to nalidixic acid, amoxicillin and ciprofloxacin were alarmingly high. Third generation cephalosporins were seen to be moderately active except against E. coli. Parasites were identified in 312 (4.78%) cases. Giardia intestinalis, Ascaris lumbricoides and Entamoeba histolytica were identified in 2.27%, 1.15% and 0.64% cases respectively. CONCLUSION: Analysis of recent nationwide studies revealed V. cholerae was the most common bacterial/parasitic agent of diarrhea across all populations, being followed by diarrheagenic E. coli and Giardia intestinalis. Periodic laboratory monitoring of antimicrobial susceptibility pattern is essential, as is formulation of effective antibiotic use policy.

Draft Genome of Escherichia coli O146 Isolate from Maulana Azad Medical College, New Delhi, India.

Here, we report the draft genome sequence of enteropathogenic Escherichia coli (EPEC) O146 strain isolated from a 1-year-old child with acute diarrhea in Delhi who recovered completely. The multidrug transporter (mdtABCD) gene, responsible for drug resistance, is present. The strain also contains the astA gene, an additional virulence determinant.

A comparative study of conventional and molecular techniques in diagnosis of campylobacter gastroenteritis in children.

BACKGROUND: Campylobacter species are a significant cause of gastroenteritis among children worldwide. Conventional methods for detection of Campylobacter spp. based on cultural isolation and biochemical tests are cumbersome and time consuming. Because of their superior sensitivity and cost effectiveness, molecular methods are often used for identification of the pathogens. AIMS: To evaluate different diagnostic methods for identification of Campylobacter. MATERIALS AND METHODS: Faecal samples were collected from 585 children (age ≤ 12 years) with acute diarrhoea admitted in a tertiary-care hospital, excluding children already on antimicrobial therapy. All samples were examined by four methods: Grams' staining, culture methods, Enzyme-Immuno Assay, and Polymerase Chain Reaction (PCR). After Grams' staining, samples were inoculated on modified charcoal cefoperazone deoxycholate agar. ProSpecT™ Microplate Assay® and PCR assay using cadF gene was done for detection of Campylobacter specific antigen and DNA, respectively, in faecal samples. McNemar's test was used to compare the results wherever applicable. RESULTS: 197 cases (33.67%) were found to be positive for Campylobacter by at least one method. But only 121 (20.78%) out of the 585 stool specimens tested fulfilled the positivity criteria, i.e., positive either by culture or by any two tests among other three. Culture had very low sensitivity (37.19%), whereas PCR had the highest (96.69%) sensitivity but lowest positive predictive value (86.03%). Rapid Grams' staining technique (sensitivity 63.64%) was found to be better than culture. Detection by PCR and ELISA was significantly better than by culture on selective media and Grams' staining (p<0.0001). CONCLUSIONS: Molecular techniques significantly increased detection rates of Campylobacter in children with diarrhoea. However, enzyme-immuno assay with high accuracy has the advantage of applicability in resource-poor settings.

A comparison of nested PCR assay with conventional techniques for diagnosis of intestinal cryptosporidiosis in AIDS cases from northern India.

Cryptosporidiosis is a very important opportunistic infection and is responsible for significant morbidity and mortality in HIV/AIDS patients. Although current laboratory methods are generally considered adequate to detect high concentrations of oocysts, they fail to detect cases of cryptosporidiosis in many immunocompromised patients. The present study was done to determine the diagnostic efficacy of modified Ziehl-Neelsen (ZN), antigen detection ELISA, and a nested PCR assay for detection of Cryptosporidium in 58 adult AIDS cases with diarrhea from the ART clinic of Lok Nayak Hospital, New Delhi. Cryptosporidium was detected in 17 (29.4%), 39 (67.3%), and 45 (77.5%) cases by modified ZN staining, antigen ELISA, and nested PCR assay, respectively. Taking nested PCR as the gold standard, specificity of both modified ZN staining and Cryptosporidium antigen detection ELISA was 100% while the sensitivity of the tests was 37.8% and 86.6%, respectively. PCR was more sensitive than the other two diagnostic modalities but required a more hands-on time per sample and was more expensive than microscopy. PCR, however, was very adaptable to batch analysis, reducing the costs considerably. This assay can therefore have considerable advantages in the treatment of immunosuppressed individuals like AIDS patients, allowing their early diagnosis and decreasing the morbidity and the mortality.

Enteric pathogens, immune status and therapeutic response in diarrhea in HIV/AIDS adult subjects from north India.

Intestinal infection causing diarrheal disease is a dominant contributor to high morbidity and mortality in developing countries. This intervention study aimed to assess the response of specific anti-microbial and anti-retroviral therapy (ART) on enteropathogens identified in HIV/AIDS adult subjects from northern India. Seventy five ART naive (group 1) and seventy five ART adherent (group 2) HIV/AIDS adult subjects with diarrhea were enrolled. Stool samples from all subjects were examined for enteropathogens by wet mount, staining methods, culture and ELISA. Subjects with enteropathogens were started on specific therapy as per National AIDS Control Organisation, Government of India's guidelines. Follow-up stool samples were examined after 2-4 weeks of completion of therapy for persistence/clearing of enteropathogens. CD4+ T lymphocyte count was done for all subjects. At enrollment, group 1 had 26.13% bacterial, 57.66% parasitic & 16.22% fungal pathogens while group 2 had 11.9%, 69.05% & 19.05% pathogens, respectively. Parasitic diarrhea was more common than bacterial diarrhea. The coccidian parasites (Cryptosporidium spp. & Isospora belli) were the common parasites identified. Clearance of enteric pathogens was significant after specific anti-microbial therapy (p = 0.0001). Persistence of enteropathogens was seen primarily for coccidian parasites. Clearance of enteropathogens after specific therapy and the diagnostic yield of stool specimens were influenced by the CD4+ counts. Immune competence coupled with specific anti-microbial therapy displays the best response against enteric pathogens.

Single and multiple liver abscesses in adults in Delhi are amoebic in origin: a clinical and microbiological study.

Single and multiple liver abscesses in Delhi are predominantly amoebic and must be distinguished from pyogenic abscesses which frequently require drainage. Mixed abscesses are larger, harbouring Gram negative rods. Multiple abscesses are not always pyogenic and presence of bacteria does not imply a primary pyogenic source.

Use of dried blood blotted on filter paper to detect dengue IgM antibody and dengue NS1 antigen.

A cross sectional study was conducted at the Department of Microbiology, Maulana Azad Medical College, New Delhi, to determine the efficacy of capillary blood samples collected on filter paper compared with serum samples to detect dengue IgM antibody and dengue NS1 antigen. The serum and capillary blood samples were collected from 104 suspected cases of dengue fever at Lok Nayak Hospital, India. The blood eluted from the filter paper and the serum samples were tested for dengue IgM antibody and dengue NS1 antigen by commercial capture ELISA kits. Of the 104 patients, samples from 61 patients tested positive for dengue IgM antibodies with both the serum and filter paper. Samples from 38 patients were negative for both serum and filter paper. Five samples showed discordant results. The filter paper method had sensitivity of 96.8% and specificity of 97.4% for dengue IgM antibody compared to the serum samples. The positive predictive value (PPV) and negative predictive value (NPV) for the filter paper to detect dengue IgM were 95.31% and 95.0%, respectively. The Kappa value (>0.80) showed agreement between the filter paper and serum results for IgM antibody detection. NS1 antigen was detected 28 serum and 28 filter paper samples and was not detected in 72 serum and 72 filter paper samples. Discordant results were seen in 4 samples. The filter paper method had a sensitivity of 96.5% and a specificity of 96.0% compared to the serum for detecting dengue NS1 antigen. The PPV and NPV for the filter paper samples in detecting dengue NS1 antigen were 90.3% and 98.7%, respectively. The Kappa value showed agreement (>0.80) between the serum and filter paper results for detecting dengue NS1 antigen. The results show filter paper samples are a reasonable alternative to serum for detecting dengue infecting.

Increasing antimicrobial resistance of campylobacter jejuni isolated from paediatric diarrhea cases in a tertiary care hospital of new delhi, India.

INTRODUCTION: The resistance to the clinically important antimicrobial agents, particularly the fluoroquinolones and the macrolides, is increasing among the Campylobacter isolates. Only limited data is available regarding the changing antimicrobial resistance pattern in the Indian scenario. METHODOLOGY: Three hundred fifty cases (ages ≤12years) of acute diarrhoea, who were admitted to a tertiary-care hospital, were investigated for Campylobacter spp. The antimicrobial susceptibilities of all the C. jejuni isolates were assessed by the disk diffusion method according to the CLSI guidelines. RESULTS: A total of 36 isolates of C. jejuni were tested for their antimicrobial susceptibilities. A high degree of resistance to the fluoroquinolones (100% to Nalidixic acid and 86.66% to Ciprofloxacin) was detected in the Campylobacter isolates. The frequency of resistance against Tetracycline was 33.33% and that against Erythromycin was 22.2%. Fifteen (41.66%) isolates were multiresistant, being resistant to 3 or more antimicrobial agents. CONCLUSIONS: An increased resistance to the quinolones and the macrolides and multidrug resistance warrant a reconsideration of their use as the drugs of choice in patients with severe gastroenteritis when Campylobacter is the presumed cause.

Clinical and Microbiological Profile of HIV/AIDS Cases with Diarrhea in North India.

Intestinal infections are a significant cause of morbidity and mortality in people living with HIV/AIDS (PLWHA) especially in developing countries. The present study was conducted to assess the clinical and microbiological spectrum in HIV/AIDS cases with diarrhea and to correlate the occurrence of such pathogens with stool characters, HIV seropositivity status, and CD4 counts. Stools from 154 HIV seropositive subjects and 50 HIV negative controls were examined by direct microscopy, fecal cultures, and serological tests (Clostridium difficile Toxin A, Cryptosporidium antigen, and Entamoeba histolytica antigen ELISA). CD4 T cell enumeration was done using FACS count (Becton Dickinson). The study showed a male preponderance (112 males and 42 females). Weakness, abdominal pain, and anorexia were the most common symptoms. Coccidian parasites were the most common cause of diarrhea in HIV seropositive cases. C. parvum was seen in 60.42% while Isospora belli in 9.03%. Amongst the bacterial pathogens C. difficile was detected in 18.06%, diarrheagenic Escherichia coli in 11.11%, and Shigella spp. in 2.78%. Pathogen isolation rates were more in HIV seropositive cases and subjects with low CD4 T lymphocyte counts. Regular monitoring of CD4 T lymphocyte counts and screening for enteric pathogens will help improve the quality of life for PLWHA.

An unusual presentation of Salmonella typhi bacteriuria in a 10 year old girl from North India.

Enteric fever caused mainly by Salmonella typhi (S. typhi), has a high incidence in India. We report a case of enteric fever in a ten year old patient presenting with clinical history of less than one week. Although blood culture, the most important diagnostic modality in early stage of enteric fever was sterile, S. typhi was isolated from culture of urine sample. Serum antibody titres against S. typhi were unusually raised considering that the infection was still in its early stage. Intravenous ceftriaxone therapy was given leading to complete recovery.

Epidemiology of opportunistic infections and its correlation with CD4 T-lymphocyte counts and plasma viral load among HIV-positive patients at a tertiary care hospital in India.

The study was conducted to find the correlation of CD4 counts and plasma viral load (PVL) with opportunistic infections (OIs) in HIV-positive patients. A total of 43 drug-naive patients enrolled in the study. Absolute CD4 counts and PVL were measured. On the basis of symptoms, sputum, stool, and blood samples were obtained for laboratory tests. Oral swabs were obtained from all the patients. Pneumocystis jiroveci pneumonia was found in 45.2% patients (odds ratio [OR] = 12.8 for CD4 counts 4.0 log(10) copies/mL). Pulmonary tuberculosis (TB; OR = 8.0 for PVL >4.0 log(10) copies/mL) and streptococcal pneumonia (detected only with CD4 counts <50 cells/mm( 3) and PVL >4.0 log(10) copies/mL) were seen in 41.9% and 12.9% patients, respectively. Among patients with diarrhea, Giardia lamblia was detected in 31% patients (OR = 3.0 for CD4 counts 4.0 log(10) copies/mL) and Cryptosporidium in 17.2% patients (OR = 1.8 for CD4 counts 4.0 log(10) copies/mL). Shigellosis and Clostridium difficile toxin was present in 13.6% patients and 6.8% patients, respectively.

Enteric Pathogens in HIV/AIDS from a Tertiary Care Hospital.

BACKGROUND: Patterns of enteric infections in HIV in developing countries may differ in several important ways from developed countries, the knowledge of which can often guide therapy when resource limitations hamper the exact diagnosis of the etiological agent in HIV-associated diarrhea. OBJECTIVES: The primary objective of this study was to define and compare the microbial etiologies of diarrhea in HIV-1 infected and non infected patients and in HIV infected non diarrheal patients. MATERIALS AND METHODS: This study was conducted between April 2007 and July 2007 at the Department of Microbiology, Maulana Azad Medical College, New Delhi. Stool samples from 50 HIV seropositive cases with diarrhea (study group), 50 HIV seropositive cases without diarrhea (control group I), and 50 HIV seronegative cases with diarrhea (control group II) were examined. After the diagnosis of HIV infection was made, routine parasitological and bacteriological detection was done. An ELISA was used for the detection of Clostridium difficile toxin and Cryptosporidium antigen in stool samples. RESULTS: The overall prevalence of enteric parasitosis in the study group was 20% and the bacteria identified were Escherischia coli in 24% of the case, Clostridium difficile in 10% of the cases, Salmonella species and Vibrio cholerae in 4% of the cases, and Shigella species in 2% of the cases. Candida species was identified in 36% of the cases. CONCLUSIONS: Identification of the etiological agent of diarrhea in a patient with AIDS is very important as it can help in the institution of appropriate therapy and the reduction of morbidity and mortality in these patients.

Diarrhoeal outbreak of Vibrio cholerae 01 Inaba in Delhi.

V. cholerae O1 Eltor serotype Ogawa has been causing most of the cholera outbreaks in India till recently. However this communication reports the occurrence of Vibrio Cholerae O1 Inaba in Delhi in 2005, as a predominant causative organism of cholera in children. All strains isolated were sensitive to gentamicin and a high level of resistance towards nalidixic acid and amoxicillin was seen. There was no case fatality.