February
fosfomycin in pus may be reached with a significant or critical delay in some abscesses. However, prolonged half-life and sustained high concentration have been achieved with multiple doses in some patients. Although overall tissue distribution is good, the clinical response depends on the location of pus/abscess cavities as well. Fosfomycin has excellent tissue penetration properties, which may be attributed to its low molecular weight, low plasma protein binding, high hydrophilicity, and a good volume of distribution. Despite favourable tissue penetration of fosfomycin, monotherapy with fosfomycin has limited use because of the rapid development of resistance and so it is advised to use fosfomycin with other antimicrobials.[7] However, fosfomycin single time oral therapy has been recommended in uncomplicated urinary tract infection.[8] Thus the present study emphasizes the high prevalence of MDR P. aeruginosa producing -lactamase enzymes of diverse mechanisms. Fosfomycin MICs as shown are still less in India and so for treating abscesses combination of fosfomycin with other antimicrobials represents good option as it shows high-tissue penetration in soft- tissue infections.
5.
6.
7.
8.
2. 3.
4.
Antimicrobial susceptibility of multidrug resistant Gram negative bacteria to fosfomycin. Eur J Clin Microbiol Infect Dis 2008;27:439-43. Anuilar MD, Morosini MI, Campo RD, Castillo MG, Zamora J, Canton R. In vitro activity of fosfomycin against a collection of clinical Pseudomonas aeruginosa isolates from 16 Spanish Hospitals: Establishing the validity of Standard Broth Microdilution as Susceptibility Testing Method. Antimicrob Agents Chemother 2013;57:5701-3. Okazaki M, Suzuki K, Asano N, Araki K, Shukuya N, Egami T, et al. Effectiveness of fosfomycin combined with other antimicrobial agents against multidrug resistant Pseudomonas aeruginosa isolates using the efficacy time index assay. J Infect Chemother 2002;8:37-42. Sauermann R, Karch R, Langenberger H, Kettenbach J, Mayer_Helm B, Petsch M, et al. Antibiotic abcess penetration: Fosfomycin levels measured in pus and simulated Concentration-Time profiles. Antimicrob Agents Chemother 2005;49:4448-54. Gupta V, Rani H, Singla N, Kaistha N, Chander J. Determination of Extended-Spectrum β-Lactamases and AmpC production in uropathogenic isolates of escherichia coli and susceptibility to fosfomycin. J Lab Physicians 2013;5:90-3.
R Garg, *V Gupta, J Chander, M Kaur Department of Microbiology, Government Medical College Hospital, Chandigarh, India
References 1.
S161
Correspondence
Tam VH, Chang KT, Abdelraouf K, Brioso CG, Ameka M, McCaskey LA, et al. Prevalence, resistance mechanisms, and susceptibility of multidrug-resistant bloodstream isolates of Pseudomonas aeruginosa. Antimicrob Agents Chemother 2010;54:1160-4. Dinh A, Salomon J, Bru JP, Bernard L. Fosfomycin: Efficacy against infections caused by multidrug-resistant bacteria. Scand J Infect Dis 2012;44:182-9. Chislett RJ, White G, Hills T, Turner DP. Fosfomycin susceptibility among extended-spectrum-beta-lactamaseproducing Escherichia coli in Nottingham, UK. J Antimicrob Chemother 2010;65:1076-7. Falagas ME, Kanellopoulou MD, Karageorgopoulas DE, Dimopoulos G, Rafailidis PI, Skarmoutsou ND, et al.
*Corresponding author (email: ) Received: 07-03-2013 Accepted: 09-10-2014 Access this article online Quick Response Code:
Website: www.ijmm.org PMID: *** DOI: 10.4103/0255-0857.150954
Detection and distribution pattern of prevalent genotypes of Hepatitis-C virus among chronic hepatitis patients from Kumaon region of Uttarakhand, India Dear Editor, Hepatitis C virus (HCV) has population-specific genotypes and provides valuable epidemiological and therapeutic information. Hence, the importance of genotype knowledge is high for clinicians in devising therapeutic strategies. Till date, no scientific data is available on the
confirmation of genotypic distribution of HCV in the Kumaon region of Uttarakhand. In this study, 39 serum samples positive for anti-HCV antibodies using 4th generation HCV TRI-DOT (J. Mitra and Co. Pvt. Ltd., New Delhi) were further processed using nested RT-PCR and restriction fragment length
www.ijmm.org
S162
Indian Journal of Medical Microbiology
polymorphism (RFLP)-based genotyping. Eleven representative samples from 33 confirmed positives in PCR/RFLP were subjected for sequencing (SciGenom Labs Pvt. Ltd., Kerala). To simplify the genotype and subtype, a phylogenetic tree was constructed using MEGA 5.05 software.[1] The approval of ethical committee was obtained prior to the study, and informed consent was obtained from individual patient.
vol. 33, Supplement 1
On comparing the Uttarakhand HCV isolates with different countries HCV isolates, seven of the HCV-3a Indian isolates showed clustering with the Canadian
RNA was extracted from 150 l of serum by using Nucleo-pore RNA Sure Virus Kit (Genetix Biotech Asia Pvt. Ltd., New Delhi). Reverse transcription polymerase chain reaction (RT-PCR) was performed following the method described previously.[2] HCV genotyping was performed by the RFLP method as described by Pour et al.[3] In the present study, out of 39 serum samples positive for anti-HCV antibodies by 4th Generation TRI-DOT Immunoassay, 33 were found positive by RT-PCR assay, showing a detection rate 84.6%. The RFLP patterns (genotype 3a: 129 bp, 145 bp, 99 bp; genotype 3b: 97 bp, 145 bp, 99 bp) reported earlier[2,3] using the same sets of restriction enzymes were different compared to the present study [Table 1]. The cogency of restrictions patterns that we found in this study were further confirmed by in silico digestion of sequenced PCR amplicons with the same restriction enzymes using MB advanced DNA analysis v6.84. This difference may be due to mutation at the recognition site of Apa I due to which tube-A showed a 145-bp band instead of 129 bp (3a) or 97 bp (3b). The sequence-based HCV genotyping and subtyping revealed predominance of HCV 3a genotype (72.72%) followed by 3b (18.18%) in the State. Studies from India suggest north–south divide, with predominance of HCV genotype 3 in the Northern states while genotype 1 in the Southern states.[4] In the present study, genotype 3 was found in all 33 patients based on restriction patterns and/or sequencing. Table 1: Restriction pattern observed for HCV-3a and HCV-3b isolates Genotype Total sample Nested RT-PCR no=33 amplicon (174 bp) Tube A Tube B Tube C (bp) (bp) (bp) 145 145 99 HCV-3a N=27# 145 145 129 N=2* 174+145 174+145 129 N=1** HCV-3b 174 174 99 N=1*** N=2 (untypable by RFLP) Eleven samples were selected for sequencing (#5 randomly selected samples,* and **samples were identified as genotype 3a and sample*** and 2 untypable by RFLP as genotype 3b)
Figure 1: Phylogenetic tree shown is based on retrieved sequences of 5′UTR of HCV from different countries. HCV isolates from Uttarakhand region are highlighted with solid black dots. The alignment was done with Clustal W method, and neighbour-joining statistical method was used for constructing the phylogenetic tree
www.ijmm.org
February
HCV-3a isolates and sequence homology of more than 85% for all isolates [Figure 1]. Similarly, the remaining three HCV-3b isolates from Uttarakhand also clustered with Canadian HCV-3b isolates with more than 86% identity at nucleotide level for all isolates [Figure 1]. The Uttarakhand HCV isolates when compared with each other were found to have more sequence similarities except one isolate (HCV-3a/UKD-2/India) which showed lower sequence identity with other HCV 3a isolates.
2.
In our study, out of 33 patients who were HCV positive by PCR assay, 28 (84.8%) were from Udham Singh Nagar (USN) district and the remaining 5 (15.2%) were from the Nainital District. In our previous study from this region on blood donors, significantly higher HCV seropositivity was seen among the Sikh population from the USN district.[5] USN is largely inhabited by the Sikh community, where many people are found to be associated in some form of working or visiting Canada. It is hypothesised that this could be the reason for the close association of Indian and Canadian HCV isolates. However, a larger cohort from this geographical region is required to test the hypothesis that this genotype may have spread from Canada to USN place via population migration and admixture.
5.
Acknowledgments
3. 4.
Bukh J, Purcell RH, Miller RH. Importance of primer selection for the detection of hepatitis C virus RNA with the polymerase chain reaction assay. Proc Natl Acad Sci U S A 1992;89:187-91. Pour MH, Keivani H, Sabahi F, Alavian SM. Determination of HCV Genotypes, in Iran by PCR-RFLP. Iran J Public Health 2006;35:54-61. Chakravarti A, Ashraf A, Malik S. A study of changing trends of prevalence and genotypic distribution of hepatitis C virus among high risk groups in North India. Indian J Med Microbiol 2013;31:354-9. Rawat V, Bhatt U, Singhai M, Kumar A, Malik YP. Prevalence of hepatitis C virus infection among blood donors of Kumaon region of Uttarakhand. Indian J Med Microbiol 2013;31:313-4.
YPS Malik, N Kumar, *V Rawat, K Sharma, A Kumar, M Singhai, M Kumar Division of Biological Standardization (YPSM), Indian Veterinary Research Institute (YPSM, NK, KS), Izatnagar, Bareilly, Uttar Pradesh, Department of Microbiology (VR, MS, MK), Department of Medicine (AK), Government Medical College, Haldwani, Uttarakhand, India *Corresponding author (email: ) Received: 21-08-2013 Accepted: 30-07-2014
The authors are thankful to the Director, Indian Veterinary Research Institute (IVRI) for providing funds to carry out the research work.
Access this article online Quick Response Code:
References 1.
S163
Correspondence
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 2011;28:2731-9.
Website: www.ijmm.org PMID: *** DOI: 10.4103/0255-0857.148838
Mucor-culture/molecular analysis necessary for identification Dear Editor, This is with reference to a case report by Angeline, et al.,[1] ‘Subcutaneous Mucor Zygomycosis with potential life threatening visceral complication’, published in the Indian J Med Microbiol 2013;31 (2):182-4. We have some reservations regarding use of the word ‘Mucor’ in the title. The authors have described a case of subcutaneous zygomycosis. Taxonomic organisation of zygomycetes has been described in a review by Ribes et al.,[2] and in greater detail by Hoffman et al.[3] Mucor is one of
the 57 genera of the order—Mucorales of the phylum Zygomycota.[3] The findings in the case report included involvement of subcutaneous site and presence of eosinophilic granuloma that is Splendore Hoeppli phenomenon. These findings are commonly seen in infections due to Entomophthorales but rarely with Mucorales.[2] Response to fluconazole therapy has been described before for Entomophthorales[4] whereas resistance has been reported consistently against Mucorales.[5] Though the patient
www.ijmm.org
Copyright of Indian Journal of Medical Microbiology is the property of Medknow Publications & Media Pvt. Ltd. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
fosfomycin in pus may be reached with a significant or critical delay in some abscesses. However, prolonged half-life and sustained high concentration have been achieved with multiple doses in some patients. Although overall tissue distribution is good, the clinical response depends on the location of pus/abscess cavities as well. Fosfomycin has excellent tissue penetration properties, which may be attributed to its low molecular weight, low plasma protein binding, high hydrophilicity, and a good volume of distribution. Despite favourable tissue penetration of fosfomycin, monotherapy with fosfomycin has limited use because of the rapid development of resistance and so it is advised to use fosfomycin with other antimicrobials.[7] However, fosfomycin single time oral therapy has been recommended in uncomplicated urinary tract infection.[8] Thus the present study emphasizes the high prevalence of MDR P. aeruginosa producing -lactamase enzymes of diverse mechanisms. Fosfomycin MICs as shown are still less in India and so for treating abscesses combination of fosfomycin with other antimicrobials represents good option as it shows high-tissue penetration in soft- tissue infections.
5.
6.
7.
8.
2. 3.
4.
Antimicrobial susceptibility of multidrug resistant Gram negative bacteria to fosfomycin. Eur J Clin Microbiol Infect Dis 2008;27:439-43. Anuilar MD, Morosini MI, Campo RD, Castillo MG, Zamora J, Canton R. In vitro activity of fosfomycin against a collection of clinical Pseudomonas aeruginosa isolates from 16 Spanish Hospitals: Establishing the validity of Standard Broth Microdilution as Susceptibility Testing Method. Antimicrob Agents Chemother 2013;57:5701-3. Okazaki M, Suzuki K, Asano N, Araki K, Shukuya N, Egami T, et al. Effectiveness of fosfomycin combined with other antimicrobial agents against multidrug resistant Pseudomonas aeruginosa isolates using the efficacy time index assay. J Infect Chemother 2002;8:37-42. Sauermann R, Karch R, Langenberger H, Kettenbach J, Mayer_Helm B, Petsch M, et al. Antibiotic abcess penetration: Fosfomycin levels measured in pus and simulated Concentration-Time profiles. Antimicrob Agents Chemother 2005;49:4448-54. Gupta V, Rani H, Singla N, Kaistha N, Chander J. Determination of Extended-Spectrum β-Lactamases and AmpC production in uropathogenic isolates of escherichia coli and susceptibility to fosfomycin. J Lab Physicians 2013;5:90-3.
R Garg, *V Gupta, J Chander, M Kaur Department of Microbiology, Government Medical College Hospital, Chandigarh, India
References 1.
S161
Correspondence
Tam VH, Chang KT, Abdelraouf K, Brioso CG, Ameka M, McCaskey LA, et al. Prevalence, resistance mechanisms, and susceptibility of multidrug-resistant bloodstream isolates of Pseudomonas aeruginosa. Antimicrob Agents Chemother 2010;54:1160-4. Dinh A, Salomon J, Bru JP, Bernard L. Fosfomycin: Efficacy against infections caused by multidrug-resistant bacteria. Scand J Infect Dis 2012;44:182-9. Chislett RJ, White G, Hills T, Turner DP. Fosfomycin susceptibility among extended-spectrum-beta-lactamaseproducing Escherichia coli in Nottingham, UK. J Antimicrob Chemother 2010;65:1076-7. Falagas ME, Kanellopoulou MD, Karageorgopoulas DE, Dimopoulos G, Rafailidis PI, Skarmoutsou ND, et al.
*Corresponding author (email: ) Received: 07-03-2013 Accepted: 09-10-2014 Access this article online Quick Response Code:
Website: www.ijmm.org PMID: *** DOI: 10.4103/0255-0857.150954
Detection and distribution pattern of prevalent genotypes of Hepatitis-C virus among chronic hepatitis patients from Kumaon region of Uttarakhand, India Dear Editor, Hepatitis C virus (HCV) has population-specific genotypes and provides valuable epidemiological and therapeutic information. Hence, the importance of genotype knowledge is high for clinicians in devising therapeutic strategies. Till date, no scientific data is available on the
confirmation of genotypic distribution of HCV in the Kumaon region of Uttarakhand. In this study, 39 serum samples positive for anti-HCV antibodies using 4th generation HCV TRI-DOT (J. Mitra and Co. Pvt. Ltd., New Delhi) were further processed using nested RT-PCR and restriction fragment length
www.ijmm.org
S162
Indian Journal of Medical Microbiology
polymorphism (RFLP)-based genotyping. Eleven representative samples from 33 confirmed positives in PCR/RFLP were subjected for sequencing (SciGenom Labs Pvt. Ltd., Kerala). To simplify the genotype and subtype, a phylogenetic tree was constructed using MEGA 5.05 software.[1] The approval of ethical committee was obtained prior to the study, and informed consent was obtained from individual patient.
vol. 33, Supplement 1
On comparing the Uttarakhand HCV isolates with different countries HCV isolates, seven of the HCV-3a Indian isolates showed clustering with the Canadian
RNA was extracted from 150 l of serum by using Nucleo-pore RNA Sure Virus Kit (Genetix Biotech Asia Pvt. Ltd., New Delhi). Reverse transcription polymerase chain reaction (RT-PCR) was performed following the method described previously.[2] HCV genotyping was performed by the RFLP method as described by Pour et al.[3] In the present study, out of 39 serum samples positive for anti-HCV antibodies by 4th Generation TRI-DOT Immunoassay, 33 were found positive by RT-PCR assay, showing a detection rate 84.6%. The RFLP patterns (genotype 3a: 129 bp, 145 bp, 99 bp; genotype 3b: 97 bp, 145 bp, 99 bp) reported earlier[2,3] using the same sets of restriction enzymes were different compared to the present study [Table 1]. The cogency of restrictions patterns that we found in this study were further confirmed by in silico digestion of sequenced PCR amplicons with the same restriction enzymes using MB advanced DNA analysis v6.84. This difference may be due to mutation at the recognition site of Apa I due to which tube-A showed a 145-bp band instead of 129 bp (3a) or 97 bp (3b). The sequence-based HCV genotyping and subtyping revealed predominance of HCV 3a genotype (72.72%) followed by 3b (18.18%) in the State. Studies from India suggest north–south divide, with predominance of HCV genotype 3 in the Northern states while genotype 1 in the Southern states.[4] In the present study, genotype 3 was found in all 33 patients based on restriction patterns and/or sequencing. Table 1: Restriction pattern observed for HCV-3a and HCV-3b isolates Genotype Total sample Nested RT-PCR no=33 amplicon (174 bp) Tube A Tube B Tube C (bp) (bp) (bp) 145 145 99 HCV-3a N=27# 145 145 129 N=2* 174+145 174+145 129 N=1** HCV-3b 174 174 99 N=1*** N=2 (untypable by RFLP) Eleven samples were selected for sequencing (#5 randomly selected samples,* and **samples were identified as genotype 3a and sample*** and 2 untypable by RFLP as genotype 3b)
Figure 1: Phylogenetic tree shown is based on retrieved sequences of 5′UTR of HCV from different countries. HCV isolates from Uttarakhand region are highlighted with solid black dots. The alignment was done with Clustal W method, and neighbour-joining statistical method was used for constructing the phylogenetic tree
www.ijmm.org
February
HCV-3a isolates and sequence homology of more than 85% for all isolates [Figure 1]. Similarly, the remaining three HCV-3b isolates from Uttarakhand also clustered with Canadian HCV-3b isolates with more than 86% identity at nucleotide level for all isolates [Figure 1]. The Uttarakhand HCV isolates when compared with each other were found to have more sequence similarities except one isolate (HCV-3a/UKD-2/India) which showed lower sequence identity with other HCV 3a isolates.
2.
In our study, out of 33 patients who were HCV positive by PCR assay, 28 (84.8%) were from Udham Singh Nagar (USN) district and the remaining 5 (15.2%) were from the Nainital District. In our previous study from this region on blood donors, significantly higher HCV seropositivity was seen among the Sikh population from the USN district.[5] USN is largely inhabited by the Sikh community, where many people are found to be associated in some form of working or visiting Canada. It is hypothesised that this could be the reason for the close association of Indian and Canadian HCV isolates. However, a larger cohort from this geographical region is required to test the hypothesis that this genotype may have spread from Canada to USN place via population migration and admixture.
5.
Acknowledgments
3. 4.
Bukh J, Purcell RH, Miller RH. Importance of primer selection for the detection of hepatitis C virus RNA with the polymerase chain reaction assay. Proc Natl Acad Sci U S A 1992;89:187-91. Pour MH, Keivani H, Sabahi F, Alavian SM. Determination of HCV Genotypes, in Iran by PCR-RFLP. Iran J Public Health 2006;35:54-61. Chakravarti A, Ashraf A, Malik S. A study of changing trends of prevalence and genotypic distribution of hepatitis C virus among high risk groups in North India. Indian J Med Microbiol 2013;31:354-9. Rawat V, Bhatt U, Singhai M, Kumar A, Malik YP. Prevalence of hepatitis C virus infection among blood donors of Kumaon region of Uttarakhand. Indian J Med Microbiol 2013;31:313-4.
YPS Malik, N Kumar, *V Rawat, K Sharma, A Kumar, M Singhai, M Kumar Division of Biological Standardization (YPSM), Indian Veterinary Research Institute (YPSM, NK, KS), Izatnagar, Bareilly, Uttar Pradesh, Department of Microbiology (VR, MS, MK), Department of Medicine (AK), Government Medical College, Haldwani, Uttarakhand, India *Corresponding author (email: ) Received: 21-08-2013 Accepted: 30-07-2014
The authors are thankful to the Director, Indian Veterinary Research Institute (IVRI) for providing funds to carry out the research work.
Access this article online Quick Response Code:
References 1.
S163
Correspondence
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 2011;28:2731-9.
Website: www.ijmm.org PMID: *** DOI: 10.4103/0255-0857.148838
Mucor-culture/molecular analysis necessary for identification Dear Editor, This is with reference to a case report by Angeline, et al.,[1] ‘Subcutaneous Mucor Zygomycosis with potential life threatening visceral complication’, published in the Indian J Med Microbiol 2013;31 (2):182-4. We have some reservations regarding use of the word ‘Mucor’ in the title. The authors have described a case of subcutaneous zygomycosis. Taxonomic organisation of zygomycetes has been described in a review by Ribes et al.,[2] and in greater detail by Hoffman et al.[3] Mucor is one of
the 57 genera of the order—Mucorales of the phylum Zygomycota.[3] The findings in the case report included involvement of subcutaneous site and presence of eosinophilic granuloma that is Splendore Hoeppli phenomenon. These findings are commonly seen in infections due to Entomophthorales but rarely with Mucorales.[2] Response to fluconazole therapy has been described before for Entomophthorales[4] whereas resistance has been reported consistently against Mucorales.[5] Though the patient
www.ijmm.org
Copyright of Indian Journal of Medical Microbiology is the property of Medknow Publications & Media Pvt. Ltd. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
