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   2009| April-June  | Volume 27 | Issue 2  
    Online since April 21, 2009

 
 
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ORIGINAL ARTICLES
Molecular typing of methicillin-resistant Staphylococcus aureus strains by PCR-RFLP of SPA gene: A reference laboratory perspective
PL Mehndiratta, P Bhalla, A Ahmed, YD Sharma
April-June 2009, 27(2):116-122
DOI:10.4103/0255-0857.45363  PMID:19384033
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.
  21 9,013 896
BRIEF COMMUNICATIONS
Factors affecting the nasal carriage of methicillin-resistant Staphylococcus aureus in human immunodeficiency virus-infected patients
J Chacko, M Kuruvila, GK Bhat
April-June 2009, 27(2):146-148
DOI:10.4103/0255-0857.49429  PMID:19384039
Human immunodeficiency virus-infected patients attending skin outpatient department were studied for nasal carriage of methicillin-resistant Staphylococcus aureus (MRSA) and associated factors affecting nasal colonization. Nasal swabs were used for isolation of S. aureus. MRSA were detected by agar screen and agar dilution methods. Careful examination for dermatoses was carried out. Forty-six of the 60 (76.67%) outpatients with HIV infection were colonized with S. aureus in the anterior nares. Significant number of S. aureus carriers were in the 31-40 year age group. Methicillin resistance was found in eight (17.39%) isolates. Of the 46 S. aureus strains, 29 (63%) were resistant to erythromycin, 69.5% to co-trimoxazole and 41.3% to ciprofloxacin. Co-trimoxazole use was found to be a risk factor for S. aureus carriage ( P = 0.0214) but not for methicillin resistance. Hospital stay for more than 10 days was a risk factor for methicillin resistance whereas stay for more than 25 days was found to be a highly significant risk factor. Dermatophytosis and herpes simplex virus infection were other risk factors for nasal carriage of S. aureus.
  9 4,305 604
CORRESPONDENCE
Epidemiology of candida bloodstream infections in a tertiary care institute in India
A Kothari, V Sagar
April-June 2009, 27(2):171-172
DOI:10.4103/0255-0857.49440  PMID:19384050
  8 5,393 756
BRIEF COMMUNICATIONS
Leptospirosis outbreak in 2005: L.T.M.G. hospital experience
Meenakshi Mathur, Anuradha De, Dilip Turbadkar
April-June 2009, 27(2):153-155
DOI:10.4103/0255-0857.49431  PMID:19384041
Nine hundred and forty two serum samples from clinically suspected cases of leptospirosis admitted in Lokmanya Tilak Municipal General Hospital, Mumbai during July-September 2005 were tested by LeptoTek Dri-dot/Leptocheck. One hundred and sixty five positive sera by these tests were sent to I.R.R., Mumbai, for detection of leptospira IgM antibodies by ELISA (PanBio). Eighty seven positive sera were also sent to B.J. Medical College, Pune, for microscopic agglutination test (MAT) for serovar identification. Seropositivity with LeptoTek Dri-dot/Leptocheck was 34.3%. Adults and males predominated. All patients were febrile. The commonest presentation in adults was jaundice (81.4%), followed by oliguria (37.6%). In children, myalgia was commonest (75.6%), followed by conjunctival suffusion (54.7%). IgM ELISA positivity was 69.1% and MAT positivity was 29.9%. Commonest serovar detected in this geographical area was Leptospira icterohaemorrhagiae (42.9%), followed by L. bataviae, L. tarassovi, and L. pomona . Considering at least two of the above three serological tests positive, 127 cases could be diagnosed and only 89.8% of them could be diagnosed by ELISA and rapid test. Therefore, along with rapid serological tests, IgM ELISA should be routinely done for laboratory diagnosis of leptospirosis.
  7 4,024 424
ORIGINAL ARTICLES
Role of azithromycin against clinical isolates of family enterobacteriaceae: A comparison of its minimum inhibitory concentration by three different methods
N Chayani, S Tiwari, G Sarangi, B Mallick, A Mohapatra, BP Paty, P Das
April-June 2009, 27(2):107-110
DOI:10.4103/0255-0857.45361  PMID:19384031
Purpose: To determine the effect of azithromycin, a new azalide antibiotic, on clinical isolates of the family Enterobacteriaceae and to determine and compare its minimum inhibitory concentration (MIC) by disk diffusion, agar dilution and E-test methods. Materials and Methods: One hundred fifty-nine bacterial strains belonging to the family Enterobacteriaceae, isolated from different clinical samples, were tested for their susceptibility to azithromycin by disk diffusion, agar dilution and E-test methods. The MIC values were analysed and the percentages of agreement between the different methods were mentioned. Results: Of the 159 isolates of the family Enterobacteriaceae, 60.37% were E. coli followed by Klebsiella species 28.3%, Salmonella and Shigella species 3.77% and Enterobacter and Citrobacter species 1.88% each. Maximum isolates were obtained from urine 117/159 (73.58%). Azithromycin was found to be more active against Salmonella and Shigella species, showing 100% sensitivity the by E-test and 83.33% by the disk diffusion methods. In the agar dilution method, 83.33% of Salmonella and 66.66% of Shigella species were sensitive to azithromycin. The overall agreement between disk diffusion and agar dilution method was 96.8%, between agar dilution and E-test was 88% and between disk diffusion and E-test was 91.2%. Conclusion: Azithromycin may become an important addition to our antimicrobial strategies, especially for the treatment of bacterial diarrhoea and infections caused by Salmonella typhi.
  7 9,723 882
Paragonimus heterotremus infection in Nagaland: A new focus of paragonimiasis in India
TS Singh, H Sugiyama, A Umehara, S Hiese, K Khalo
April-June 2009, 27(2):123-127
DOI:10.4103/0255-0857.49424  PMID:19384034
Purpose: To determine the prevalence of paragonimiasis among the patients who were attending the tuberculosis (TB) clinics at the Community Health Centre, Pfutsero, Phek District, Nagaland. To determine the species of Paragonimus that cause infection in humans and the crustacean host that acts as the infectious source for humans. Materials and Methods: Sputum specimens were examined microscopically for Paragonimus eggs and acid fast bacilli. Blood samples were tested by microenzyme-linked immunosorbant assay for Paragonimus-specific immunoglobulin G antibodies. Crab extracts prepared by digestion with artificial gastric juice were examined for Paragonimus metacercariae under a stereoscopic microscope. The species identification of the parasite was based on morphological and molecular characterizations of eggs and metacercariae employing polymerase chain reaction and DNA sequencing. Results: Seven out of the 14 patients tested seropositive for paragonimiasis and Paragonimus eggs were detected in sputum of two out of the seven seropositive patients, indicating a prevalence of 50% and an egg detection rate of 14%, respectively. The prevalence was highest in the 10-30 year age group. More males got the infection than females, the ratio being 5:2. P. heterotremus was identified as the causative agent of human paragonimiasis and Potamiscus manipurensis as the crab host. Conclusions: The study revealed that paragonimiasis has been endemic in Pfutsero, Nagaland, and half of the patients attending the TB clinic were actually suffering from pulmonary paragonimiasis. This is the first confirmed report of an endemic focus of paragonimasis and description of P. heterotremus as the causative agent in Nagaland, India.
  6 4,723 403
CASE REPORTS
Unusual presentation of entomophthoromycosis
RC Michael, JS Michael, MS Mathews, V Rupa
April-June 2009, 27(2):156-158
DOI:10.4103/0255-0857.49432  PMID:19384042
Rhinoentomophthoromycosis caused by Conidiobolus sp commonly presents as a chronic granulomatous lesion that affects the rhinofacial subcutaneous tissue. We present an 18-year-old girl who presented with progressive bilateral proptosis and loss of vision since 2 weeks. Biopsy and fungal cultures confirmed diagnosis of Conidiobolus sp infection of the paranasal sinuses bilaterally with orbital extension and blindness. The clinical picture was complicated by the presence of sputum-positive cavitatory pulmonary tuberculosis, which was diagnosed at the same time. To our knowledge, this is the first such case to be reported from India. We also discuss the management of entomophthoromycosis. Despite many reports of success, there remains no consensus on the treatment of Conidiobolus infections of the nose and the paranasal sinuses with antifungal agents.
  5 4,238 366
BRIEF COMMUNICATIONS
Stacking gels: A method for maximising output for pulsed-field gel electrophoresis
See Kah Heng, Chua Kek Heng, SD Puthucheary
April-June 2009, 27(2):142-145
DOI:10.4103/0255-0857.49428  PMID:19384038
Pulsed field gel electrophoresis (PFGE), the gold standard of molecular typing methods, has a major disadvantage of an unusually long electrophoretic time. From the original protocol of 6 days, it was modified to 3 days and subsequently to a single day. We describe the procedure of stacking five to six gels one on top of another in order to increase and maximize the output in a shorter time without compromising the resolution and reproducibility. All the variables that affect pulsed field gels during electrophoresis were taken into consideration. We firstly optimized the parameters to be used and secondly determined whether stacking of five to six gels had any effect on the molecular separation during electrophoresis in comparison with a single gel run. DNA preparation, restriction, electrophoresis, staining and gel documentation was carried out based on previously published methods. Gels were analysed using BioNumerics and dice coefficient and unweighted pair group methods were used to generate dendrograms based on 1.5% tolerance values. Identical band profiles and band resolution-separation were seen in the PFGE patterns with single gel and multiple stacking gels. Cluster analysis further strengthened the fact that results from stacking gels were reproducible and comparable with a single gel run. This method of stacking gels saves time and maximizes the output at the same time. The run time for a single gel was about 28 hours, but with six stacked gels the run time was 54 hours compared with 28 × 6 = 168 hours if they were run separately as single gels thus saving time of 67.86%. Beside the big factor of saving time, stacking gels save resources (electricity, reagents, water, chemicals and working time) by increasing the sample throughput in a shorter time without compromising on quality of data. But optimization of working parameters is vital depending on the PFGE system used.
  3 4,157 298
ORIGINAL ARTICLES
Quantitation of hepatitis B virus DNA in plasma using a sensitive cost-effective "in-house" real-time PCR assay
Hubert Darius J Daniel, John G Fletcher, George M Chandy, Priya Abraham
April-June 2009, 27(2):111-115
DOI:10.4103/0255-0857.45362  PMID:19384032
Background: Sensitive nucleic acid testing for the detection and accurate quantitation of hepatitis B virus (HBV) is necessary to reduce transmission through blood and blood products and for monitoring patients on antiviral therapy. The aim of this study is to standardize an "in-house" real-time HBV polymerase chain reaction (PCR) for accurate quantitation and screening of HBV. Materials and Methods: The "in-house" real-time assay was compared with a commercial assay using 30 chronically infected individuals and 70 blood donors who are negative for hepatitis B surface antigen, hepatitis C virus (HCV) antibody and human immunodeficiency virus (HIV) antibody. Further, 30 HBV-genotyped samples were tested to evaluate the "in-house" assay's capacity to detect genotypes prevalent among individuals attending this tertiary care hospital. Results: The lower limit of detection of this "in-house" HBV real-time PCR was assessed against the WHO international standard and found to be 50 IU/mL. The interassay and intra-assay coefficient of variation (CV) of this "in-house" assay ranged from 1.4% to 9.4% and 0.0% to 2.3%, respectively. Virus loads as estimated with this "in-house" HBV real-time assay correlated well with the commercial artus HBV RG PCR assay ( r = 0.95, P < 0.0001). Conclusion: This assay can be used for the detection and accurate quantitation of HBV viral loads in plasma samples. This assay can be employed for the screening of blood donations and can potentially be adapted to a multiplex format for simultaneous detection of HBV, HIV and HCV to reduce the cost of testing in blood banks.
  3 8,583 846
Identification of lysine positive non-fermenting gram negative bacilli (Stenotrophomonas maltophilia and Burkholderia cepacia complex)
V Gautam, P Ray, P Vandamme, SS Chatterjee, A Das, K Sharma, S Rana, RK Garg, SK Madhup, M Mahajan, M Sharma
April-June 2009, 27(2):128-133
DOI:10.4103/0255-0857.49425  PMID:19384035
Background: The Burkholderia cepacia complex (BCC) and Stenotrophomonas maltophilia are closely related groups of non-fermenting gram-negative bacilli (NFGNBs) having a similar spectrum of infections ranging from superficial to deep-seated and disseminated infections. Identification of these lysine decarboxylase-positive NFGNBs lags behind in most Indian laboratories. A simplified identification scheme was devised for these two pathogens that allowed us to isolate them with an increasing frequency at our tertiary care institute. Materials and Methods: A simple five-tube conventional biochemical identification of these bacteria has been standardized. In the beginning, some of the isolates were confirmed from the International B. cepacia Working group, Belgium. Molecular identification and typing using recA polymerase chain reaction-restriction fragment length polymorphism was also standardized for BCC. For short-term preservation of BCC, an innovative method of preserving the bacteria in Robertson's cooked medium tubes kept in a domestic refrigerator was developed. Results: Thirty-nine isolates of BCC isolates were obtained from various specimens (30 from blood cultures) and 22 S. maltophilia (13 blood cultures and 9 respiratory isolates) were isolated during the year 2007 alone. Conclusions: BCC and S. maltophilia can be identified with relative ease using a small battery of biochemical reactions. Use of simplified methods will allow greater recognition of their pathogenic potential and correct antimicrobials should be advised in other clinical laboratories and hospitals.
  3 8,980 772
CORRESPONDENCE
Seroprevalence of human immunodeficiency virus and syphilis in blood donors of Delhi
R Ekadashi, S Langer
April-June 2009, 27(2):167-168
DOI:10.4103/0255-0857.49437  PMID:19384047
  2 2,485 307
Immunosuppression level in HIV-positive patients with oropharyngeal candidiasis
Usha Arora, Maninder Jagdev, Neerja Jindal
April-June 2009, 27(2):174-175
DOI:10.4103/0255-0857.49442  PMID:19384052
  2 2,685 323
GUEST EDITORIAL
Laboratory microbiology to clinical microbiology: Are we ready for a transition?
Sanjay Bhattacharya
April-June 2009, 27(2):97-99
DOI:10.4103/0255-0857.49422  PMID:19384029
  2 5,267 840
BRIEF COMMUNICATIONS
Detection of porcine rotavirus from tissue and faecal specimens
Suji Prabha, Susan Verghese
April-June 2009, 27(2):149-152
DOI:10.4103/0255-0857.49430  PMID:19384040
Porcine small intestinal sub-mucosa is a cell-free collagen matrix that has demonstrated its ability as a scaffold material. Transplantation poses special hazards because grafted tissues and organs transmit pathogens efficiently, especially viruses. Rotavirus is thought to be confined to the intestine, causing acute diarrhoea. The purpose of this study was to evaluate the porcine intestinal tissue scaffold for Rotavirus and to study the incidence of this virus among pig herds. Only one isolate was successfully adapted to grow in cell line MA 104 from faecal samples. This isolate was further confirmed by reverse transcriptase polymerase chain reaction and sequence analysis.
  1 5,417 385
CASE REPORTS
Diagnosis of spontaneous bacterial peritonitis: Role of tween 80 and triton X in ascitic fluid cultures
RN Iyer, D Kapoor
April-June 2009, 27(2):159-161
DOI:10.4103/0255-0857.49433  PMID:19384043
A patient with alcoholic cirrhosis of the liver, portal hypertension with hepatic encephalopathy and spontaneous bacterial peritonitis (SBP) was admitted in an obtunded condition. Attempts at delineating the aetiology of the SBP using conventional cultures as well as automated systems were not successful. The use of non-anionic surfactant agents such as Tween 80-incorporated blood agar and Triton X treatment of the specimens facilitated the growth of Klebsiella pneumoniae from the ascitic fluid, which otherwise would have been concluded to represent culture-negative neutrocytic ascites. Thus, the use of the aforementioned agents could be explored in elucidating the aetiology of body cavity infections when conventional methods fail.
  1 4,476 384
CORRESPONDENCE
Nocardia puris endophthalmitis
D Papaventsis, N Siafakas, L Kondyli, M Akritidou, P Pantazi, E Perdikari, G Bethimoutis, G Chatzakis, L Zerva
April-June 2009, 27(2):168-170
DOI:10.4103/0255-0857.49438  PMID:19384048
  1 2,569 163
Combination of three rapid tests: An alternative approach to confirmatory laboratory diagnosis of HIV infection in Bangladesh
SU Munshi, J Ahmed, M Ahmed, A Nessa, S Tabassum
April-June 2009, 27(2):170-171
DOI:10.4103/0255-0857.49439  PMID:19384049
  1 3,332 289
Cryptosporidial oocysts in gastric aspirate of an infant
H Rani, V Gupta, N Gulati, J Chander
April-June 2009, 27(2):172-174
DOI:10.4103/0255-0857.49441  PMID:19384051
  1 2,510 228
ORIGINAL ARTICLES
Clinical evaluation of the mycobacteriophage-based assay in rapid detection of Mycobacterium tuberculosis in respiratory specimens
S Prakash, SK Katiyar, S Purwar, JP Singh
April-June 2009, 27(2):134-138
DOI:10.4103/0255-0857.49426  PMID:19384036
Context: Search for a cost-effective, rapid and accurate test has renewed interest in mycobacteriophage as a tool in the diagnosis of tuberculosis (TB). There has been no reported data on the performance of phage assay in a high burden, low-resource setting like Kanpur city, India. Aims: To assess the sensitivity and specificity of the FASTPlaque TB™ kit ability to impact the bacillary load in the phage assay and its performance in the sputum smear sample negative cases. Materials and Methods: The study involved a cross-sectional blinded assessment of phage assay using the FASTPlaque TB™ kit on 68 suspected cases of pulmonary TB against sputum smear microscopy by Ziehl-Neilsen staining and culture by the LJ method. Results: The sensitivity, specificity and positive and negative predictive values of the phage assay were 90.7, 96, 97.5 and 85.7%, respectively. The assay was negative in all the five specimens growing mycobacteria other than TB. The sensitivity of the phage assay tended to decrease with the bacillary load. Of the smear-negative cases, three were false negative, and all of which were detected by the phage assay. Smear microscopy (three smears per patient) had a sensitivity and specificity of 93 and 64%, respectively. Conclusions: The phage assay has the potential clinical utility as a simple means of rapid and accurate detection of live Mycobacterium tuberculosis bacilli; however, its performance has been inconsistent across various studies, which highlights that the assay requires a high degree of quality control demanding infrastructure and its performance is vulnerable to common adversities observed in "out of research" practice settings like storage, transport and cross-contamination.
  1 5,326 517
REVIEW ARTICLE
Histopathology for the diagnosis of infectious diseases
E Gupta, P Bhalla, N Khurana, T Singh
April-June 2009, 27(2):100-106
DOI:10.4103/0255-0857.49423  PMID:19384030
Histopathological examination of tissue biopsies for the identification of infectious organisms is a very important diagnostic tool. Conventional culture confirmation of tissue biopsies often fail to identify any pathogen as, first of all, invariably most of the tissue samples that are collected and sent for culture isolation are inappropriately collected in formalin, which prevents pathogen growth in culture media. Inadequate processing like grinding, etc. further hinders isolation. Presence of inhibitors like dead tissue debris, fibers, etc. also delays isolation. Microbiologists often lack expertise in identifying infectious pathogens directly from tissue biopsies by microscopic visualization. This review therefore acquaints microbiologists with the various methods available for detecting infectious agents by using histological stains. On histopathological examination of the tissue biopsy once, it is determined that a disease is likely to be due to an infection and has characterized the inflammatory response and hence associated microorganisms should be thoroughly looked for. Although some microorganisms or their cytopathic effects may be clearly visible on routine haematoxylin- and eosin-stained sections, additional histochemical stains are often needed for their complete characterization. Highly specific molecular techniques, such as immunohistochemistry, in situ hybridization and nucleic acid amplification, may be needed in certain instances to establish the diagnosis of infection. Through appropriate morphologic diagnoses and interlaboratory communication and collaboration, direct microscopic visualization of tissue samples can thus be very helpful in reaching a correct and rapid diagnosis.
  1 17,296 1,270
BRIEF COMMUNICATIONS
Seroprevalence of hepatitis E virus immunoglobulin G and M antibodies in adults: A hospital-based study
K Bashir, N Hussain, S Hasnain, S Elahi
April-June 2009, 27(2):139-141
DOI:10.4103/0255-0857.49427  PMID:19384037
Sporadic cases of Hepatitis E virus (HEV) infection occur throughout the year in Pakistan. The aim of this study was to determine the prevalence of HEV immunoglobulin (Ig) G and IgM antibodies in 93 hepatitis B and C-negative patients as such patients are not routinely tested further despite having signs and symptoms of hepatitis. Anti-HEV IgG and IgM were detected by the enzyme-linked immunosorbant assay technique. Among them five patients (5.4%) were positive for HEV IgG and IgM, with an average age of 30.95 ± 15.35 years. Hepatitis E infection was independent of the sex. Liver function tests of hepatitis E-positive IgG and IgM patients showed increased values of serum glutamate oxaloacetate transaminase, serum glutamate pyruvate transaminase, alkaline phosphatase and bilirubin that indicate damaged hapatocytes and malfunctioning of the liver.
  - 3,528 383
CASE REPORTS
Pulmonary botryomycosis in a patient with down syndrome
FA Al-Rabee, WA Hayajneh, M Shorman, R Al-Hubail
April-June 2009, 27(2):161-163
DOI:10.4103/0255-0857.49434  PMID:19384044
Pulmonary botryomycosis is a rare chronic, pyogranulomatous infection affecting the lung parenchyma. We describe here the clinical and histopathological findings of pulmonary botryomycosis reported for the first time in a Down syndrome female who required prolonged intensive care. This case has other different unique aspects. It is the first case to present with empyema, the second case involving the right lower lobe and the first case managed by decortication.
  - 2,789 208
Post-traumatic osteomyelitis due to aeromonas species
L Gunasekaran, S Ambalkar, RA Samarji, A Qamruddin
April-June 2009, 27(2):163-165
DOI:10.4103/0255-0857.49435  PMID:19384045
We report a case of Aeromonas osteomyelitis due to injury in a sewage worker. He presented with cellulitis of the left foot. Radiographs showed evidence of osteomyelitis involving the head and neck of the fifth metatarsal. Aeromonas species was isolated from the tissue and swab from the foot. The head and neck of the fifth metatarsal were excised and the patient improved on 4 weeks of intravenous meropenem followed by 4 weeks of oral clindamycin and ciprofloxacin.
  - 3,706 255
CORRESPONDENCE
Circulating phage type of Vibrio cholerae in Mysore
S Srirangaraj, D Venkatesha
April-June 2009, 27(2):166-167
DOI:10.4103/0255-0857.49436  PMID:19384046
  - 2,295 289
RESEARCH SNIPPETS
Research snippets from the medical world
P Desikan
April-June 2009, 27(2):176-177
DOI:10.4103/0255-0857.49443  
  - 1,741 205

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