The prevalence of Mycoplasma hominis in Outpatients at a Tertiary Care Hospital in East India.

Introduction Mycoplasma hominis (M. hominis) is the first Mycoplasma isolated from humans in the year 1937. Though regarded as a commensal of the urogenital tract, it has been implicated in various genital and extra-genital infections namely bacterial vaginosis, cervicitis, pelvic inflammatory diseases, pyelonephritis, premature rupture of the membrane in pregnancy, infertility, sepsis in newborns, etc. The pathogenesis, prevalence, and epidemiology of genital mycoplasmas in general and M. hominis in particular in Indian women have been studied very minimally. This study aimed to study the prevalence of M. hominis carriage among symptomatic and asymptomatic sexually active women attending to the outpatient department of a tertiary care hospital in East India with or without clinically suspected genitourinary infections and to compare the detection of M. hominis by polymerase chain reaction (PCR) as compared to that of culture. Methods In this observational study, sterile Dacron swabs were used to collect two samples each from the genitourinary tract of 110 sexually active women aged 15-45 years (80 cases and 30 control). One sample was inoculated in mycoplasma broth for culture. The other was used for PCR to detect the presence of the M. hominis gene. Results Culture positivity for mycoplasma was seen in 4/80 (5%) patients clinically suspected of genitourinary infection (cases) based on their presenting signs and symptoms. In those without such suspicion (control), all cultures were negative (p=0.021). PCR was positive for M. hominis in 22 (20%) samples. Considering the PCR as the gold standard the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of culture are found to be 18.18%, 100%, 100%, and 88.25% respectively. The highest prevalence of M. hominis was in the age group 20-24 years (9/24) and 50% of all detections (11/22) were among 25-29 years. Detections were more frequent among patients with multiparity, multiple sexual partners, intrauterine contraceptive devices, lower socioeconomic status, and lower educational status. Conclusion Our study results showed that the presence of M. hominis is significantly higher in cases than in the control group. The study also indicates the need for continued research on this bacterium both in patients with genital symptoms and in asymptomatic patients.

Identification of population of bacteria from culture negative surgical site infection patients using molecular tool.

BACKGROUND: Managing surgical site infections, with negative culture report in routine diagnosis is a common dilemma in microbiology accounting more than 30% worldwide. The present study attempted to identify the presence of bacterial spp. if any in wound aspirates/swabs of culture negative surgical site infections of hospitalised patients using molecular tools. METHODS: Ninety-seven patients with post-operative SSI whose wound swabs/aspirate were negative in the conventional aerobic culture after 72 h of incubation were analysed by 16S rRNA gene specific broad range PCR. The amplified DNA fragments were sequenced by Sanger DNA sequencing method and homology of the sequence were matched using NCBI BLAST (NCBI, USA) RESULTS: Of the 97 patients, 16S rRNA based broad range PCR assay could identify the presence of bacterial pathogen in 53(54.63%) cases, of which 29 isolates were supposed to be of viable but non-culturable bacteria (VBNC), 07 were of obligatory anaerobes and 13 were of unculturable bacteria, 04 were with poly bacterial infections. CONCLUSIONS: Our study highlights the usefulness of PCR assay in detecting the presence of any VBNC, anaerobes and unculturable bacteria in SSI patients regardless of how well the bacteria may or may not grow in culture. Measures should be taken to use anaerobic culture system and PCR diagnosis along with conventional culture to detect the VBNC and unculturable bacteria where Gram stain is positive for better patient care.

Etiology of childhood diarrhoea among under five children and molecular analysis of antibiotic resistance in isolated enteric bacterial pathogens from a tertiary care hospital, Eastern Odisha, India.

BACKGROUND: Although, India has made steady progress in reducing deaths in children younger than 5 years, the proportional mortality accounted by diarrhoeal diseases still remains high. The present hospital based cross sectional study was carried out to understand the prevalence of various bacterial pathogens associated with the diarrhoea cases in under 5 years age group. METHODS: During, 1st September, 2015 to 30th November 2017, all the childhood diarrhoea cases (≤5 yrs) of SCB Medical College in Odisha, India were included in the study. Stool samples were collected and processed for the isolation of causative bacterial pathogen and the isolated bacterial pathogens were subjected to antibiotic sensitivity testing, molecular analysis of drug resistance. Clinical and demographic data were collected and analyzed. RESULTS: Three hundred twenty patients were enrolled in the study during the study period from whom 82 bacterial isolates were obtained indicating a proportional causality of 25.6% for bacterial diarrhoea among children in this region. Entero toxigenic E.coli (ETEC) accounted for majority of the cases and and more than 50% of the strains were found to be multi-drug resistant (resistant to more than 3 class of antibiotics). More than 50% of the strains were resistant to current choice of treatment like ciprofloxacin, ofloxacin and ceftriaxone and 2.4% being resistant to Imipenem. ESBL production was also observed in some of the strains and one isolate harboured the NDM-1 gene. Fluoroquinolone resistance was found to be linked with multiple mutations in the QRDR region followed by PMQR determinants. CONCLUSION: The current study, to the best of our knowledge is first of its kind which demonstrated the etiology of bacterial diarrhoea in children less than 5 years old and identified diarrheogenic E. coli as the predominant enteropathogen in Odisha. Majority of the isolates being multi-drug resistance calls for a continuous surveillance system in the region which will be helpfulin identifying emerging resistance pattern and for developing suitable intervention stategies.

Genetic diversity of Indian Plasmodium vivax isolates based on the analysis of PvMSP3ß polymorphic marker.

BACKGROUND: Malaria is one of the major communicable diseases in India and worldwide. PvMSP3ß is a highly polymorphic gene due to its large insertions and deletions in the central alanine-rich region, which, in turn, makes it a valuable marker for population genetic analysis. Very few studies are available from India about the genetic diversity of Plasmodium vivax based on PvMSP3ß gene, and hence, this study was designed to understand the molecular diversity of the P. vivax malaria parasite. The accumulating epidemiological data provide insights into the circulating genetic variants of P. vivax in India, and ultimately benefits the vaccine development. MATERIALS AND METHODS: A total of 268 samples confirmed to be positive by microscopy, rapid diagnostic test, and quantitative buffy coat test were collected from four different regions of India (Puducherry, Mangaluru, Jodhpur, and Cuttack) in the present study. Polymerase chain reaction (PCR)-based diagnosis was carried out to confirm the P. vivax monoinfection, and only the mono-infected samples were subjected to PvMSP3ß gene amplification and further restriction fragment length polymorphism (RFLP) to determine suballeles. RESULTS: Based on the size of the amplified fragment, the PvMSP3ß gene was apportioned into two major types, namely Type A genotype (1.6-2 Kb) was predominantly present in 148 isolates and Type B (1-1.5 Kb) was observed in 110 isolates. The percentage of mixed infections by PCR was 3.73%. All the PCR products were subjected to RFLP to categorize into suballeles and we detected 39 suballeles (A1-A39) in Type A, and 23 suballeles (B1-B23) in Type B genotype. A high degree of diversity was observed among the isolates collected from Mangaluru region when compared to isolates collected from other regions. CONCLUSION: The present study showed a high degree of genetic diversity of PvMSP3ß gene among the isolates collected from various parts of India. High polymorphism in PvMSP3ß gene makes it a promising marker for epidemiological and vaccine development studies.

Polymorphisms in genes associated with drug resistance of Plasmodium vivax in India.

Malaria is a sterning public health concern in India and contribute to a major part of malaria burden in Southeast Asia. Being more populated and diverse geographic conditions makes more suitable place for sustaining malaria parasite in India. Anti-malarial resistance is a major concern in the battle against malaria, and the identified molecular markers will aid us to monitor the drug resistance in endemic areas. The aim of the current study is to determine the genotype of drug resistance associated genes pvmdr-1 and pvcrt-o from four different regions of India. Especially from Puducherry and Jodhpur, there were no prior studies focused on screening of drug resistance genes in P. vivax parasite. A total of 240 positive P. vivax infected patient samples were collected from four tertiary care hospitals from four different regions of India, namely, Puducherry (PDY), Mangaluru (MAQ), Cuttack (CTC), Jodhpur (JDH). All samples were screened by microscopy, RDT, QBC, and further DNA was extracted and vivax mono-infection was confirmed by nested PCR. Randomly selected amplicons were further subjected to nucleotide sequencing. The prevalence of K10 insertion in pvcrt-o gene was detected with 18.8% in PDY, 12.5% in MAQ and 6.3% in CTC P. vivax isolates, whereas no change in nucleotide was identified in P. vivax isolates collected from JDH region. Based on the F1076L mutation in pvmdr-1 gene, resistant P. vivax isolates was highly predominant in both the regions, JDH and CTC, with 100%, followed by MAQ with 93.3% and PDY with 73.3%. This study showed less frequency of pvcrt-o and high frequency of pvmdr-1 gene variants associated with CQ resistance, which act as an indicator and the onset of P. vivax drug resistance trend in four different regions of India. Due to the poor phenotypic studies available for P. vivax parasite, the present study data for CQ resistance based on pvcrt-o and pvmdr-1 markers should assist by providing base-line data for future monitoring of drug resistance.

High Prevalence of Biofilm-Forming MRSA in the Conjunctival Flora in Chronic Dacryocystitis.

OBJECTIVES: To report the microbiological spectrum of conjunctival flora and prevalence of biofilm-forming Methicillin-resistant Staphylococcus aureus (MRSA) in conjunctival flora in chronic dacryocystitis. DESIGN: Prospective, case-control study. METHODS: We included patients with unilateral chronic dacryocystitis, and their unaffected eyes as control. Microbiological profile and antibiotic susceptibility of the isolates was determined by standard microbiological procedures. S. aureus isolates were further evaluated for Methicillin resistance by Oxacillin resistance screening agar method and mecA polymerase chain reaction (PCR) and for biofilm synthesis by Congo red agar method, Microtitre plate (MTP) assay, and ica A and ica D PCR. RESULTS: We found 95 patients with unilateral chronic dacryocystitis. Aerobic Gram-positive isolates (74.2%, n = 72) were more than Gram-negative (25.7%, n = 25) or anaerobic isolates (20.5%, n = 25). S. aureus was most common (46.4%, n = 45), followed by Pseudomonas aeruginosa (10.3%, n = 10). Gram-positive isolates showed highest sensitivity to Linezolid (100%) and higher generation fluoroquinolones. Gram-negative isolates showed good sensitivity (>90%) to all tested antibiotics. S. aureus isolates showed MRSA prevalence as 93.5% and 96.9% by Oxacillin resistance screening agar method and mecA PCR, respectively. Biofilm formation was found in 71.8% MRSA isolates by MTP assay and 58.1% MRSA isolates were resistant to ≥3 classes of antibiotics. CONCLUSIONS: Gram-positive organisms, specifically S. aureus, are the major etiological agent in chronic dacryocystitis. There is high prevalence of MRSA in these isolates and concurrent biofilm formation.

Bacteriological profile of ventilator-associated pneumonia in a tertiary care hospital.

BACKGROUND: Ventilator-associated pneumonia (VAP) is the most frequent intensive care unit (ICU)-acquired infection. The etiology of VAP and their antimicrobial susceptibility pattern varies with different patient populations and types of ICUs. MATERIALS AND METHODS: An observational cross-sectional study was performed over a period of 2 years in a tertiary care hospital to determine the various etiological agents causing VAP and to detect the presence of multidrug-resistant (MDR) pathogens in these VAP patients. Combination disk method, Modified Hodge test, ethylenediaminetetraacetic acid disk synergy test, and AmpC disk test were performed for the detection of extended-spectrum beta-lactamase (ESBL), carbapenemases, metallo-beta-lactamases (MBL), and AmpC beta-lactamases, respectively. RESULTS: The prevalence of VAP was 35%. Enterobacteriaceae (66.66%) and Staphylococcus aureus (20%) were common in early-onset VAP, while nonfermenters (50%) and Enterobacteriaceae (40.61%) were predominant from late-onset VAP. Nearly 60.87% of the bacterial pathogens were MDR. ESBL was produced by 21.74% of Enterobacteriaceae. AmpC ß-lactamase was positive in 35.29% nonfermenters and 26.08% Enterobacteriaceae. MBL was positive in 17.64% nonfermenters and 17.39% Enterobacteriaceae. Among the S. aureus isolates, 75% were cefoxitin resistant. Prior antibiotic therapy (P = 0.001) and hospitalization of 5 days or more (P = 0.001) were independent risk factors for VAP by MDR pathogens. polymyxin B, tigecycline, and vancomycin were the most sensitive drugs for Gram-negative and positive isolates respectively from VAP. STATISTICAL ANALYSIS: SPSS for Windows Version SPSS 17.0 (SPSS Inc., Chicago, IL, USA) and Chi-square with Yates correction. CONCLUSION: Late-onset VAP is increasingly associated with MDR pathogens. Treatment with polymyxin B, tigecycline, and vancomycin should be kept as last-line reserve drugs against most of the MDR pathogens.

Detection of inducible and constitutive clindamycin resistance among Staphylococcus aureus isolates in a tertiary care hospital, Eastern India.

INTRODUCTION: Clindamycin is an excellent drug for skin and soft tissue Staphylococcus aureus infections, but resistance mediated by inducible macrolide-lincosamide-streptogramin B (iMLSB) phenotype leads to in vivo therapeutic failure even though they may be in vitro susceptible in Kirby-Bauer disk diffusion method. OBJECTIVE: The study was aimed to detect the prevalence of iMLSB phenotype among S. aureus isolates by double disk approximation test (D-test) in a tertiary care hospital, Eastern India. MATERIALS AND METHODS: A total of 209 consecutive S. aureus isolates were identified by conventional methods and subjected to antimicrobial susceptibility testing by Kirby-Bauer disk diffusion method. Erythromycin-resistant isolates were tested for D-test. RESULTS: From 1282 clinical specimens, 209 nonrepeated S. aureus isolates were obtained. Majority of isolates 129 (61.7%) were methicillin-resistant S. aureus (MRSA). There was statistically significant difference between outpatients 60.1% and inpatients 39.9% (P < 0.0001). From 209 S. aureus isolates, 46 (22%) were D-test positive (iMLSB phenotype), 41 (19.6%) were D-test negative (methicillin sensitive [MS] phenotype), and 37 (17.7%) were constitutively resistant (constitutive macrolide-lincosamide-streptogramin B phenotype). The incidence of inducible, constitutive, and MS phenotype was higher in MRSA isolates compared to MS S. aureus (MSSA). The constitutive clindamycin resistance difference between MSSA and MRSA isolates were found to be statistically significant (P = 0.0086). CONCLUSION: The study revealed 22% of S. aureus isolates were inducible clindamycin resistant, which could be easily misidentified as clindamycin susceptible in Kirby-Bauer disk diffusion method. Therefore, clinical microbiology laboratory should routinely perform D-test in all clinically isolated S. aureus to guide clinicians for the appropriate use of clindamycin.

Evaluation of Genotypic and Phenotypic Methods for Detection of Methicillin Resistant Staphylococcus aureus in a Tertiary Care Hospital of Eastern Odisha.

INTRODUCTION: Methicillin resistant Staphylococcus aureus has emerged as an important pathogen in nosocomial and community acquired infections. Accurate and rapid identification of MRSA in clinical specimens is essential for timely decision of effective antimicrobial chemotherapy. AIM: The present study was conducted to compare efficacy of four conventional phenotypic methods, with mec- A based polymerase chain reaction (PCR) for MRSA identification. MATERIALS AND METHODS: Methicillin resistance was determined in 200 S.aureus isolates by oxacillin disc diffusion, cefoxitin disc diffusion, Oxacillin Resistance Screening Agar and E-test. The results were compared with mec-A based PCR. RESULTS: Among 200 S.aureus isolates 62 (31%) were positive for mec-A gene by PCR. Cefoxitin disc diffusion, Oxacillin Resistance Screening Agar and E-test showed 100% specificity. Oxacillin disc diffusion had lowest sensitivity (82.5%) and specificity (98.5%) among all. The conventional methods take more time than PCR for diagnosing MRSA. Linezolid, Vancomycin & Dalfopristin were the highly sensitive drugs against MRSA isolates. CONCLUSION: Cefoxitin disc diffusion, is rapid, simple and cheaper, hence can be used routinely as an alternative to PCR for detection of MRSA in resource constraint laboratories.

Study of biofilm in bacteria from water pipelines.

CONTEXT: A biofilm is a layer of microorganisms contained in a matrix (slime layer), which forms on surfaces in contact with water. Their presence in drinking water pipe networks can be responsible for a wide range of water quality and operational problems. AIM: To identify the bacterial isolates, obtained from water pipelines of kitchens, to evaluate the water quality & to study the biofilm producing capacity of the bacterial isolates from various sources. SETTINGS AND DESIGN: A prospective study using water samples from aqua guard & pipelines to kitchens of S.C.B Medical College hostels. MATERIALS AND METHODS: Standard biochemical procedures for bacterial identification, multiple tube culture & MPN count to evaluate water quality & tissue culture plate (TCP) method for biofilm detection was followed. STATISTICAL ANALYSIS: STATA software version 9.2 from STATA Corporation, College station road, 90 Houston, Texas was used for statistical analysis. RESULTS: One hundred eighty seven isolates were obtained from 45 water samples cultured. The isolates were Acinetobacter spp. (44), Pseudomonas spp.(41), Klebsiella spp.(36) & others . Biofilm was detected in (37) 19.78 % of the isolates (95% CI 30.08% -43.92%) including Acinetobacter spp.-10, Klebsiella spp. - 9, Pseudomonas spp. - 9, & others, majority (34) of which were from kitchen pipelines. CONCLUSION: Water from pipeline sources was unsatisfactory for consumption as the MPN counts were > 10. Most of the biofilm producers were gram negative bacilli & Pseudomonas & Acinetobacter spp. were strong (4+) biofilm producers.

NS1 Antigen Capture ELISA an Effective Method for Diagnosis of Early Dengue Infection - Report of an Outbreak at Angul District, Odisha, India.

BACKGROUND: An outbreak of dengue infection occurred in Angul district of Odisha in the month of August & September, 2011. The study was undertaken to detect NS1 antigen positivity among the study population, to compare IgM capture ELISA with NS1 antigen detection for diagnosis of dengue and to identify the predominant genotype of Dengue virus responsible for the outbreak. MATERIALS AND METHODS: Total 1020 serum samples were collected from clinically suspected cases of dengue from the outbreak. All were subjected for NS1 antigen detection, 92 were selected based on their clinical severity of illness (fever, rash, bleeding manifestation, arthralgia) for further study of IgM ELISA and platelet count and 148 NS1 positive samples were selected from different Blocks of Anugul district for RT-PCR at NIV, Pune, India. RESULTS: Five hundred and thirteen (50.2%) samples were positive for NS1 antigen (highly significant p-value <0.0001, C.I - 95%) with 88% positivity during 1-5 days. The NS1 Ag positivity was peaked to 86.9% on days 3 to 5 (Sensitivity & NPV - 100% each) & declined to 6.2% during 6-10 days with a low sensitivity of 7.14% but 100% specificity & PPV. However, the IgM antibody positivity was 81.2% on days 6 to 10 and 87.5% after 10 days (Sensitivity- 100%, Specificity-13.33%,PPV-7.14% & NPV - 100%). RT-PCR resulted 32.4% positivity (6- DEN1, 39 - DEN 2 & 3- DEN 3) among which 20% were in IgM +ve & 68% in IgM -ve cases. CONCLUSION: Therefore, early diagnosis of dengue could be mainly by NS1 antigen detection whereas Ig M ELISA is a better tool during the later stage of infection &RT-PCR is more effective in IgM -ve cases.The predominant genotype responsible for the outbreak was found to be DEN-2.

Coexistence of cutaneous tuberculosis (scrofuloderma) and hanseniasis-a rare presentation.

Cutaneous tuberculosis, pulmonary tuberculosis and hanseniasis are all caused by different spp. of Mycobacterium, an intracellular pathogen whose development depends on impaired cell mediated immunity. Scrofuloderma is the most common variant of cutaneous tuberculosis, which is characterized by a direct extension of the skin which overlies the infected lymph gland, bone or joint, that breaks down to form an undermined ulcer. We are reporting a rare association of Scrofuloderma (cutaneous tuberculosis) with Hanseniasis (leprosy) in an adult male whose immune status was controversial.

Utility of multiplex reverse transcriptase-polymerase chain reaction for diagnosis and serotypic characterization of dengue and chikungunya viruses in clinical samples.

The reemergence of chikungunya virus (CHIKV) has compounded the already existing dengue problem because of clinical similarities and common vector, demanding the need for a rapid and specific diagnosis. Thus, dengue chikungunya multiplex reverse transcriptase-polymerase chain reaction (DCmRT-PCR) was developed and validated for simultaneous detection of dengue and chikungunya viral infections and its utility in virus serotyping. Blood samples from 97 suspected dengue and chikungunya cases and 10 healthy controls were subjected to dengue and chikungunya conventional RT-PCR and DCmRT-PCR. Thirty-one of 97 samples were positive for dengue or chikungunya viral RNA by RT-PCR and DCmRT-PCR with 100% concordance. DCmRT-PCR products were cycle sequenced. Seven dengue virus strains were clustered within genotype III of DENV-3 and 4 within genotype III of DENV-1, whereas chikungunya sequences were clustered within the Central/East African genotype. DCmRT-PCR was found to be a potential rapid test for simultaneous detection of dengue and CHIKV in clinical samples along with dengue serotyping.

Intraocular Gnathostoma spinigerum: a case report.

Live intraocular nematode is a rare occurrence that is mostly reported in South East Asian countries. Herewith we report such a case from Nayagarh district of Odisha. A 28 year old female presented with swelling, redness, lacrimation, pain & diminished vision of left eye since 2 1/2 years. Slit lamp examination revealed a worm piercing iris muscle. The worm was removed by paracentesis of anterior chamber and sent to the Department of Microbiology. It was identified to be Gnathostoma spinigerum basing on the typical morphology of its cephalic end. The patient responded completely to oral albendazole therapy.

Chromobacterium violaceum: a case report of an unusual bacterium causing puerperal sepsis.

Human infections by Chromobacterium violaceum are rare. Till date 6 cases have been reported from southern and eastern parts of India. We report here a case of puerperal sepsis by C. violaceum, probably the first case from Eastern part of Orissa. The patient was successfully treated with amikacin and gatifloxacin.

Bacteremia due to Rhodococcus equi--a case report.

Rhodococcus equi (R. equi) primarily causes zoonotic infections affecting grazing animals and is an unusual cause of infection in immunocompetent human beings. We report a case of bacteremia due to R. equi a rare isolate in a child suffering from protein energy malnutrition