Preena Bhalla

Molecular typing of methicillin-resistant Staphylococcus aureus strains by PCR-RFLP of SPA gene: A reference laboratory perspective

PURPOSE To characterize methicillin-resistant Staphylococcus aureus (MRSA) strains by molecular typing based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of spa gene and to assess the utility of spa genotyping over bacteriophage typing in the discrimination of the strains. MATERIALS AND METHODS Studies were undertaken on 125 MRSA strains representing the most predominant phage types and the non phage typeable strains. Strains were typed by bacteriophage typing and PCR-RFLP of spa gene. DNA sequence analysis of the amplified spa gene fragment of the representative RFLP patterns was performed using standard protocols. RESULTS All the strains resistant to oxacillin were found to contain mec A gene. Fifty-two per cent of these strains were typeable by the international basic set of 23 phages. Five different PCR-RFLP patterns were observed among 125 MRSA strains. Non phage typeable strains were differentiated into four PCR-RFLP patterns. Sequencing of the spa gene from the representative strains of each RFLP pattern confirmed the length of these restriction fragments due to variation in the 24 bp and the 174 bp tandem repeats. It also revealed the presence of three new spa repeat patterns. CONCLUSION The study demonstrates the importance of spa genotyping in the discrimination of MRSA strains, which were otherwise indistinguishable by bacteriophage typing. spa genotyping allowed differentiation of strains within a particular phage type. Nucleotide sequencing of isolates of different PCR-RFLP patterns indicated a correlation between the RFLP patterns of a variable number of tandem repeats and the phage type. The study provides valuable information on the epidemiological characterization of MRSA strains.

Treatment of enteric fever in children on the basis of current trends of antimicrobial susceptibility of Salmonella enterica serovar typhi and paratyphi A

PURPOSE Recent reports indicate decreased susceptibility of S. typhi to fluoroquinolones, especially ciprofloxacin. Chloramphenicol has been suggested as first line therapy of enteric fever in many studies. This is a prospective study that describes the trends of antimicrobial susceptibility of S. typhi and S. paratyphi A causing bacteraemia in children and reports therapeutic failure to ciprofloxacin and evaluates the possible use of chloramphenicol, ampicillin, ciprofloxacin and third generation cephalosporins as first line therapy in the treatment of enteric fever in children. METHODS The present study was conducted from April 2004 to March 2005 in a superspeciality children hospital at New Delhi. A total of 56 S. typhi and five S. paratyphi A isolates were obtained among the 673 blood cultures performed. Antimicrobial testing was done using disk diffusion technique (NCCLS method) for 13 antimicrobials and MICs were calculated for ampicillin, ciprofloxacin, chloramphenicol and cefotaxime. Analysis of data was done using WHONET software. RESULTS All 56 isolates of S. typhi were sensitive to amoxycillin+clavulanate, gentamicin, cefixime, cefotaxime and ceftazidime. Multidrug resistance (MDR, resistance to three drugs) was seen in 22 cases (39%) and resistance to five drugs was seen in 12 cases (21%). Only two isolates were resistant to chloramphenicol (3%). MIC 90 for ampicillin, chloramphenicol, ciprofloxacin and cefotaxime were 1.0 microg/ml, 4.0 microg/ml, 64 microg/ml and 0.125 microg/ml respectively. All S. paratyphi A isolates were sensitive to ampicillin and chloramphenicol and resistant to nalidixic acid. MIC distribution data for chloramphenicol revealed elevated MIC but still in susceptible range. CONCLUSIONS There is an urgent need for further clinical studies to evaluate response to chloramphenicol in such cases. Antimicrobial susceptibility data and MIC distribution favour use of ampicillin as a drug of choice for the treatment of enteric fever. Third generation cephalosporins are also useful but their use should be restricted for complicated cases.

Typing of Methicillin resistant Staphylococcus aureus: A technical review

Increasing prevalence of Methicillin-resistant Staphylococcus aureus (MRSA) worldwide is a growing public health concern. MRSA typing is an essential component of an effective surveillance system to describe epidemiological trends and infection control strategies. Current challenges for MRSA typing are focused on selecting the most appropriate technique in terms of efficiency, reliability, ease of performance and cost involved. This review summarises the available information on application, potential and problems of various typing techniques in discriminating the strains and understanding the epidemiology of MRSA strains. The phenotypic methods in general are easier to perform, easier to interpret, cost effective and are widely available, however less discriminatory. The genotypic methods are expensive and technically demanding, however more discriminatory. Newer technologies involving sequencing of various genes are coming up as broadly applicable and high throughput typing systems. Still there is no consensus regarding the single best method for typing of MRSA strains. Phage typing is recommended as first line approach in epidemiological investigation of MRSA strains. PFGE remains the gold standard for characterisation of outbreak strains. DNA sequencing methods including MLST, spa typing, SCCmec typing and toxin gene profile typing are more practical methods for detecting evolutionary changes and transmission events. The choice of typing technique further depends on the purpose of the study, the facilities available and the utility of data generated to answer a desirable research question. A need for harmonisation of typing techniques by following standard protocols is emphasised to establish surveillance networks and facilitate global MRSA control.

A five-year survey of onychomycosis in New Delhi, India: Epidemiological and laboratory aspects

Context: The worldwide incidence of onychomycosis is increasing and it continues to spread and persist. Knowledge of the epidemiological and mycological characteristics is an important tool for control of this infection. Aims: This study seeks to improve knowledge of onychomycosis epidemiology and mycological features. Settings and Design: Over a period of five years (Jan 2000 - Dec 2005) samples from 400 patients with clinical suspected fungal nail infections, who attended dermatology out patient department at a tertiary care hospital, were obtained. Materials and Methods: 400 nail specimens of suspected onychomycosis were evaluated clinically, KOH examination and fungal culture was done. Results: Onychomycosis was present in 218 (54.5%) by culture and /or direct examination. Fingernails and toenails were infected in 65% and 32% respectively and remaining 3% had both. Conclusions: This study demonstrated that dermatophytes were main agents causing onychomycosis in our region, as well as the importance of performing direct examination and culture in diagnosis of onychomycosis.

Development of TaqMan real-time polymerase chain reaction for the detection of the newly emerging form of carbapenem resistance gene in clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii.

PURPOSE The newly emerging form of the so-called New Delhi Metallo-beta-lactamases (NDM-1) has been reported recently from patients worldwide and broadly thought as a potential source for the major global health problem. Thus, it is important to study the epidemiology of the so-called NDM-1 harbouring bacteria to prevent its further spread and to place effective control measures. The present study describes the use of the real-time polymerase chain reaction (PCR) assay for the detection of the bla NDM-1 gene using TaqMan probes among clinical isolates. MATERIALS AND METHODS Clinical isolates of Escherichia coli (11 strains), Klebsiella pneumoniae (17 strains) and Acinetobacter baumannii (six strains) that were resistant to either of the carbapenems (meropenem or imipenem) were included in the study. The presence of carbapenemases in such strains was confirmed using the modified Hodge test. A real-time PCR assay was optimized for the detection of NDM-1 using a cloned synthetic gene fragment followed by testing of the clinical isolates. The findings were further confirmed using PCR and gene sequencing. RESULTS TaqMan probe assay displayed a good detection limit with analytical sensitivity of the assay up to 10 copies of bla NDM-1 gene per reaction. The isolates of E. coli and K. pneumoniae revealed narrow range crossing point values (Cp values) between (12-17) cycles (mean Cp value 14), indicating number of bla NDM-1 gene copies of 106-108. The wider range of Cp values (15-34) cycles with a higher mean Cp value (23.6) was observed in A. baumannii with number of bla NDM-1 gene copies of 103-108. CONCLUSIONS The study demonstrates that real-time PCR assay based on TaqMan chemistry is a useful technique for the detection of bla NDM-1 harbouring clinical isolates of E. coli, K. pneumoniae and A. baumannii. The assay has great precision in measuring the number of bla NDM-1 gene copies per specimen of DNA.

PREDICTIVE FACTORS FOR SUCCESSFUL EARLY LAPAROSCOPIC CHOLECYSTECTOMY IN ACUTE CHOLECYSTITIS: A PROSPECTIVE STUDY

BACKGROUND Early laparoscopic cholecystectomy has become the treatment of choice for acute cholecystitis. However, the rate of intraoperative conversion to open surgery remains high and has provoked an interest in studying the predictive factors for better patient selection to minimize the conversion rates. MATERIALS AND METHODS 50 patients of acute cholecystitis were operated within 5 days of onset of symptoms. Comparative evaluation of the patient groups undergoing successful versus failed early laparoscopic cholecystectomy was done to identify preoperative factors predicting conversion/failure of the laparoscopic procedure. Predictive factors for intraoperative and histopathological severity of acute cholecystitis were also identified. RESULTS 40 patients underwent successful completion of early laparoscopic cholecystectomy, 8 required conversions to open, while in 2 patients the procedure had to be abandoned due to phlegmon formation. Male sex, preoperative duration of symptoms WBC counts, serum alkaline phosphatase, serum amylase, and serum C-reactive protein were significant predictors of histopathological severity of acute cholecystitis. Intraoperative and histopathological severity of acute cholecystitis had good association with conversion rate of early laparoscopic cholecystectomy. Male sex and serum C-reactive protein levels >3.6 mg/dl at admission were very strong predictors of conversion/failure of early laparoscopic cholecystectomy in acute cholecystitis. CONCLUSION Male patients of acute cholecystitis or patient with serum C-reactive protein levels of >3.6 mg/dl at admission have high risk of conversion in early laparoscopic cholecystectomy and warrant a conservative early management followed by delayed laparoscopic cholecystectomy.

Seroprevalence of hepatitis viruses in patients infected with the human immunodeficiency virus.

OBJECTIVE The co-infection of Hepatitis B and C viruses with HIV accelerates disease progression and also has an effect on the management of patients infected with HIV. The prevalence of HIV co-infection with hepatitis viruses varies widely. This study is planned to evaluate the prevalence of HIV co-infection with Hepatitis B and C viruses in North India. MATERIALS AND METHODS A total of 1178 patients enrolled in the ART center were retrospectively analyzed for the presence of HBV and HCV on the basis of the presence of HBsAg and anti-HCV markers. RESULTS In patients infected with HIV, the prevalence of co-infection with HBV was 9.9% (117/1178), the prevalence of co-infection with HCV was 6.3% (74/1178) and the prevalence co-infection with both HBV and HCV was ~1% (12/1178). DISCUSSION The prevalence rate of HBV and HCV are increasing in patients infected with HIV. Having acquired the knowledge about the importance of such a co-infection, it is essential that all the patients infected with HIV be screened for HBV and HCV co-infection.

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.

Staphylococcus aureus phage types and their correlation to antibiotic resistance

CONTEXT Staphylococcus aureus is one of the most devastating human pathogen. The organism has a differential ability to spread and cause outbreak of infections. Characterization of these strains is important to control the spread of infection in the hospitals as well as in the community. AIM To identify the currently existing phage groups of Staphylococcus aureus, their prevalence and resistance to antibiotics. MATERIALS AND METHODS Study was undertaken on 252 Staphylococcus aureus strains isolated from clinical samples. Strains were phage typed and their resistance to antibiotics was determined following standard microbiological procedures. STATISTICAL ANALYSIS Chi square test was used to compare the antibiotic susceptibility between methicillin resistant Staph. aureus (MRSA) and methicillin sensitive S. aureus (MSSA) strains. RESULTS Prevalence of MRSA and MSSA strains was found to be 29.36% and 70.65% respectively. Of these 17.56% of MRSA and 40.44% of MSSA strains were community acquired. All the MSSA strains belonging to phage type 81 from the community were sensitive to all the antibiotics tested including clindamycin and were resistant to penicillin. Forty five percent strains of phage group III and 39% of non-typable MRSA strains from the hospital were resistant to multiple antibiotics. CONCLUSION The study revealed that predominant phage group amongst MRSA strains was phage group III and amongst MSSA from the community was phage group NA (phage type 81). MSSA strains isolated from the community differed significantly from hospital strains in their phage type and antibiotic susceptibility. A good correlation was observed between community acquired strains of phage type 81 and sensitivity to gentamycin and clindamycin.

Chlamydia trachomatis causing neonatal conjunctivitis in a tertiary care center

Chlamydia trachomatis is considered a major aetiological agent of conjunctivitis in newborns. The objective of the present study was to determine the aetiology of neonatal conjunctivitis and clinico-epidemiological correlates of chlamydial ophthalmia neonatorum. Fifty-eight newborns with signs and symptoms of conjunctivitis were studied. Conjunctival specimens were subjected to Gram staining, routine bacteriological culture, culture for Neisseria gonorrhoeae and direct fluorescent antibody (DFA) staining for diagnosis of C. trachomatis infection. C. trachomatis was detected in 18 (31%) neonates. Findings suggest that since C. trachomatis is the most common cause of neonatal conjunctivitis, routine screening and treatment of genital C. trachomatis infection in pregnant women and early diagnosis and treatment of neonatal Chlamydial conjunctivitis may be considered for its prevention and control.