Luna Adhikari

High-level Aminoglycoside Resistance and Reduced Susceptibility to Vancomycin in Nosocomial Enterococci.

Objectives: The objectives of the present study were to identify the species of enterococci isolated from nosocomial infections and to determine the antibiotic susceptibility pattern with reference to high-level aminoglycosides and vancomycin. Materials and Methods: Enterococci were isolated from various clinical samples collected from patients after 72 hours of hospitalization. Various species of Enterococcus were identified by standard methods. High-level aminoglycoside resistance and vancomycin susceptibility in enterococci were detected by disk-diffusion and agar-screen methods. Results: One hundred eighty enterococcal strains were isolated from various clinical samples. Various species of Enterococcus — Enterococcus fecalis 130 (72.22%), Enterococcus casseliflavus 24 (13.33%), Enterococcus fecium 17 (9.44%), Enterococcus durans 7 (3.89%) and Enterococcus dispar 2 (1.11%) — were isolated. The highest resistance to aminoglycoside was observed among E. fecium, followed by E. durans, E. fecalis and E. casseliflavus, both by disk-diffusion and agar-screen methods. The high-level aminoglycoside resistance (HLAR) was significantly (P<0.05) higher in E. fecium by agar-screen method. All enterococci showed minimum inhibitory concentration (MIC) of ≤8 μg/mL to vancomycin. Sixteen (12.31%) E. fecalis and 3 (12.5%) E. fecium strains were intermediately resistant to vancomycin (MIC= 8 μg/mL), whereas other strains were susceptible to vancomycin. Conclusion: The occurrence of high-level aminoglycoside resistance in enterococcal isolates in our setup was high. Even though none of the enterococcal strains showed resistance to vancomycin, yet reduced susceptibility to vancomycin was noticed in our study. This would require routine testing of enterococcal isolates for HLAR and vancomycin susceptibility. Agar-screen method was found to be superior to disk-diffusion method in detecting resistant strains to aminoglycosides and vancomycin.

Evaluation of phenotypic with genotypic methods for species identification and detection of methicillin resistant in Staphylococcus aureus.

Background: Phenotypic methods for the detection of methicillin resistance are inadequate, due to presence of hetero-resistant population and dependence of environmental factors that may affect the phenotypic expression of resistance. Aims: Present study was conducted, to evaluate the efficacy of phenotypic methods for the identification of species and mec-A mediated resistance in S. aureus with polymerase chain reaction (PCR), and to assess the prevalence of the Panton-Valentine leukocidin (pvl) toxin in methicillin resistant S. aureus (MRSA) and overall S.aureus population. Materials and Methods: A total of 200 clinical isolates of Staphylococci were subjected to phenotypic and genotypic methods for the species identification and detection of MRSA. Results: The specificity and sensitivity of conventional methods in the detection of S.aureus, was found to be 100 and 97.59% respectively. However, the performance of phenotypic methods in the detection of MRSA were: Oxacillin disc diffusion (DD)-sensitivity 70.58%, specificity 75.75%; cefoxitin DD-sensitivity 86.27%, specificity 83.33%; and oxacillin agar dilution-sensitivity 92.15%, specificity 90.90%. PVL gene was detected in all mec-A positive isolates irrespective of their types. Conclusion: Phenotypic methods still preferred for the species identification, but for the reliable detection of MRSA an algorithm should include a combination of tests and apply a genotypic method for confirmation of resistance isolates showing discrepant results. Considering the high prevalence of PVL-MRSA, we recommend PCR as assay, as it has an advantage of simultaneous detection of mec-A and pvl genes by multiplex PCR.

Clinico-etiologic correlates of onychomycosis in Sikkim.

AIMS AND OBJECTIVES The etiological spectrum of any superficial mycosis is largely dependent on the flora in the immediate environment of the individual. It is influenced by the geographic, climatic and occupational factors. The study is basically to formulate baseline data for the species prevalence of various dermatophytes in patients with onychomycosis attending the Central Referral Hospital, Gangtok, Sikkim. MATERIALS AND METHODS Thirty-four clinically suspected cases of onychomycosis were subjected to mycological studies. RESULTS Thirty-two (94.12%) cases were positive for fungal elements by direct microscopy and 28 (82.35%) by culture. Young adults in the age group of 21-30 years were mainly affected. The male:female ratio was 1.125:1. Dermatophytes were isolated in 18 cases (64.29%). Trichophyton tonsurans (44.44%) was the most common isolate followed by T. mentagrophytes (22.22%), T. rubrum (11.11%), T. verrucosum (11.11%) and Microsporum audouinii (11.11%). Apart from dermatophytes, Aspergillus niger (21.43%) and Penicillium marneffei (14.28%) were also isolated. CONCLUSION Dermatophytes, mainly T. tonsurans, as well as moulds other than dermatophytes were isolated from onychomycosis patients in Gangtok, Sikkim.

Mycetoma due to Nocardia farcinica.

A 60-year-old female presented an infection due to Nocardia farcinica and demonstrated successful intervention with surgical exploration and prolonged combined chemotherapy. She was brought with a history of multiple discharging sinuses over the right foot and swelling of the same foot since two months after a thorn prick. Physical examination showed chronic skin changes on the leg, with swelling and yellow fluid-draining sinuses. She was admitted in the surgery in-patients, and under strict aseptic conditions, pus and two swabs were collected from the discharging sinuses.

Molecular characterization of community- & hospital-acquired methicillin-resistant & methicillin-sensitive Staphylococcus aureus isolates in Sikkim.

Background & objectives: The two major genotypic markers that distinguish community acquired (CA) from hospital acquired (HA) methicillin resistant Staphylococcus aureus (MRSA) isolates are the architecture of mobile genetic element (SCCmec type) and presence of panton valentine leukocidin (PVL) toxin. This study was conducted to determine the molecular characteristics of CA- and HA- MRSA and methicillin sensitive S. aureus (MSSA) isolates in Sikkim. Methods: A total of 150 clinical isolates of S. aureus isolated from various clinical specimens were subjected to duplex (mec-A and pvl gene) and multiplex (SCCmec typing) PCR. Results: Of the 150 isolates, 53 (35.33%) and 66 (44%) were positive for mec-A (MRSA) and pvl genes, respectively. Thirty eight (25.33%) met the definition of CA-MRSA and 15 (10%) of HA-MRSA and the remaining 63 (42%) and 34 (22.66%) as CA- and HA-MSSA, respectively. No significant difference was seen in the distribution of PVL toxin in MRSA and MSSA isolates, but it was significantly (P<0.001) high in overall MRSA isolates than in MSSA. The majority of the MRSA isolates showed a double amplification band of SCCmec type III plus V (54.71%), and only a fewer isolates were amplified by single DNA fragments of type I (1.88%), III (3.77%), IVa (1.88%) and V (11.32%). SCCmec types I, III, IVa, were found only in HA-MRSA isolates, whereas type V in both the CA- and HA-MRSA. AST pattern showed that 18.42 per cent (7/38) and 46.66 per cent (7/15) were multidrug resistant (MDR)-CA-MRSA and MDR-HA-MRSA, respectively. Interpretation & conclusions: The present results show that SCCmec type V MRSA has been on the rise, and genotypic markers such as pvl gene detection used for the differentiation of these clinically distinct isolates of MRSA may not be reliable.

Infectious disease markers in blood donors at Central Referral Hospital, Gangtok, Sikkim.

Sir, A safe blood is a critical component in improving health care and in preventing the spread of infectious diseases globally. Yet the quality and safety of blood transfusion is still a concern for a health-care personnel. The extent of infectious disease markers of major blood-borne pathogens [hepatitis B (HBV), hepatitis C (HCV), human immunodeficiency virus (HIV), syphilis and malarial parasite] was observed as with age and sex distribution for the donor population of the blood bank of a tertiary care hospital of Gangtok during 2001-2008. Of the 3735 donors, 2423 (64.87%) were replacement and 1312 (35.13%) voluntary donors; male donors were 3434 (91.94%) and females were 301 (8.06%) with overall seroprevalence of major blood-borne pathogens (HIV, HBV, HCV and Syphilis) was 1.63%. The sero-positivity of HIV, HBsAg, anti- HCV, and syphilis was 0.32, 0.78, 0.27 and 0.27%, respectively. Unlike other studies,[1,2] which showed a lower prevalence of HIV, HBsAg, and anti-HCV in voluntary donors, we observed a higher HIV, HBsAg, and anti-HCV sero- positivity in voluntary donors as compared to replacement donors, though the difference in prevalence in these two groups was not statistically significant [Chi-square test (with Yates correction) = 0.02974 (P = 0.10); 0.01495; (P = 0.9027); 1.738 (P = 0.1874) respectively]. Whereas syphilis positivity was found to be higher in replacement donors as compared to voluntary donors though not statistically significant [Chi-square test (with Yates correction) = 0.4513 (P = 0.5017)], which was in agreement with other studies.[1,2] HIV and anti-HCV sero-positivity was nil from 2001 to 2005 among our donors. There was no significant change in the prevalence of HIV, HBsAg, HCV, and syphilis positivity over the period (Chi - square for trend = 5.913, P = 0.0120; 0.01728, P = 0.6776; 2.440, P = 0.1183; 0.04003, P = 0.8414, respectively). There was no HIV, HCV and syphilis positive case among female donors in our study population. The HBV positivity was the only infection found in one female donor in our total female donors in the age group of 21-40, which was statistically significant (P = 0.0062). There were no HIV and syphilis positive cases below the age of 21 years. No sample showed malarial parasites. There was not a single case positive for co-infection with two or more infectious agents. Though the screening tests used for these infections were not 100% sensitive or specific,[3,4] and the approximation of the infectious disease markers in our donor population was less, there is no scope of complacency.

Susceptibility rates of Pseudomonas aeruginosa strains to quinolones

Sir, Pseudomonas aeruginosa is an important bacterial pathogen, particularly as a cause of infections in hospitalized patients and revealed trends of increasing antimicrobial resistance. However, resistance to fluoroquinolone antibiotics has been reported in recent years as well. This crosssectional study included a total of 932 clinical samples received from various departments of Central Referral Hospital, Gangtok from August 2005 to August 2008. The samples were cultured in 5% sheep blood agar and MacConkey agar at 37°C for 18−24 hours. The isolates were preliminarily identified as Pseudomonas spp. by biochemical and physiological tests, colony morphology, production of diffusible pigments, and a grape-like odour. Gram-negative bacilli with above-mentioned characteristic features were considered as P aeruginosa on the basis of a positive oxidase test, a triple sugar iron (TSI) agar reaction of alkaline over no change, growth at 42°C and production of bright-blue to blue green diffusible pigment on Mueller-Hinton agar. Nonpigmented isolates were identified as P. aeruginosa by the above-mentioned characters along with oxidation of glucose, but not disaccharides, hydrolysis of acetamide, and reduction of nitrates to nitrogen gas. One sample was collected from each patient. The susceptibility of P aeruginosa strains to ciprofloxacin (5 μg), ofloxacin (5 μg), levofloxacin (5 μg), norfloxacin (10 μg), and pefloxacin (5 μg) was studied by Kirby-Bauer disc diffusion method according to NCCLS (National Committee for Clinical and Laboratory Standards) criteria. Out of a total of 932 clinical samples received, 682 samples were pus, 125 were urine, 83 were blood, and 42 were sputum. Of them 268 (28.76%) were isolated having P aeruginosa strains [pus (196), urine (36), blood (24), and sputum (12)]. Two hundred and eight (77.61%) isolates were obtained from in-patients, while only 60 (22.39%) were from out-patients. Ciprofloxacin (47.76%) was found to be the most effective anti-pseudomonal agent followed by levofloxacin (44.78%). However, this difference was not significant (P>0.05). The susceptibility patterns of ciprofloxacin and levofloxacin were highly significant when analyzed with the susceptibility patterns of norfloxacin (23.88%), ofloxacin (20.90%), and pefloxacin (8.96%), (Chi-square: 225.75, p value <0.0001). Belgian study reported that the resistance rate of P aeruginosa to ciprofloxacin had gradually increased over years (13% in 1991, 16−18% in 1993−1995, 30% in 1998−1999).[1] Other studies from India reported ciprofloxacin resistance rate to be between 12.5% and 30.7%.[2,3] Levofloxacin resistance has been reported to be between 16.9% and 36% in different studies.[2,4] Ofloxacin resistance has been reported by other researchers to be between 19% and 62.5%.[2,5]

Recurrent dermatophytosis: A rising problem in Sikkim, a Himalayan state of India

Changing pattern of dermatophytic infection among people of Sikkim over the past few years and its recurrence rate has brought a need to do a study on clinical pattern and its recurrence from this part of the country. The objectives of this study are to discern the clinical patterns of dermatophytosis, identification of the isolated fungi to its species level and to see the pattern of its recurrence. The study was carried out from January 2015 to May 2016. A total of 192 samples were collected from the patients with clinical findings of dermatophytic infection. Required history of the patients was taken, followed by clinical examination of the lesions and sample collection. The samples were processed for mycological study till species identification and a follow up of patients were done to assess its recurrence pattern. The age distribution of the patients was from 2 to 80 years. The mean and median age was 30.33 and 33 years respectively. The male female ratio was 1.8:1. Dermatophytosis was noted more commonly in students (n = 64, 33.33%) and jawans (n = 44, 22.92%). Maximum occurrence was noted from April to July (n = 106, 55.20%) and was seen mainly in young Hindu males. Tinea corporis (n = 104, 54.16%) was the most common clinical manifestation followed by tinea unguium (n = 30, 15.63%). T. mentagrophyte (40%) was the most common species followed by T. schoenleinii (33.3%), T. tonsurans (16.6%) and T. rubrum (6.6%). The recurrence rate was seen most commonly in clinical cases of tinea faciei 100%, followed by tinea pedis 80% and tinea unguium 46.6%. Overall clinical cure rate was 58.3% and recurrence rate was 34.3%. In the isolated species of dermatophytes, the recurrence rate was 73.68% and that of non-dermatophytes it was 28.07%. Dermatophytosis is an important health problem with high recurrence in Sikkim with difference in the etiological agent from other parts of India.

Seroprevalence of Hepatitis B, Hepatitis C, and human immunodeficiency virus and their risk factors in adolescents in East Sikkim, a Himalayan state of India

Background: Sikkim is a northeastern state of India, located in the Himalayas with migratory population from neighboring states and Nepal. Although there is high prevalence of human immunodeficiency virus (HIV) and Hepatitis B and C virus (HBV and HCV) in other northeastern states of India, currently, this information is lacking in Sikkim’s adolescent population, which inspired the researchers to conduct this study. Methods: This is a cross-sectional study with 490 adolescent participants between the age group of13–20 years. Schools/colleges and the participants were randomly selected. Blood samples of selected participants were collected after their interview and were tested for antiHIV, hepatitis B surface antigen, and anti-HCV. Findings: Mean age of the participants was 16.96 years. Only 1 (0.2%) participant was positive for HBsAg, and none was positive for anti-HIV and anti-HCV. About 92 (18.78%), 90 (18.37%), 79 (16.12%), and 12 (2.45%) participants had respectively undergone tattoo/body piercing, invasive (medical) procedure, shared personal items, and had received blood transfusion but none of them provided information on their use of injectable drugs. A total of 33 (6.73%) participants expressed their involvement in heterosexual relation, of which 29 (29/215, 13.49%) were boys and 4 (4/275, 1.45%) were girls (p<0.01). Among them, 13(13/33, 39.39%) participants expressed their involvement with multiple sexual partners and16 (16/33, 48.49%) participants had taken protective measures. None of the girls provided history about their partner taking any protective measures. Interpretation: To the best of our knowledge, this is the first study reporting on the prevalence of HIV, HBV, and HCV in the adolescent population of Sikkim. Our results indicate that there is a very low prevalence of HBV and absence of HIV and HCV infections in the studied population. However, the studied population is at risk of acquiring these infections and therefore necessary steps must be taken to prevent the transmission of these viruses. Funding: Indian Council of Medical Research.