Krishnamoorthy Gopinath

All India Institute of Medical Sciences, New Dilli, NCT, India

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Publications (24)81.71 Total impact

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    ABSTRACT: Tuberculosis is a major health problem in India, and the emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) strains of Mycobacterium tuberculosis (Mtb) has further complicated the situation. Though several studies characterizing drug sensitive and drug resistant strains are available in literature, almost all studies are done on unrelated strains. Therefore, the objective of this study was to compare the proteomic data of four sequential isolates of Mtb from a single patient who developed MDR-TB during the course of anti-tuberculosis therapy (ATT). In this study, using two-dimensional (2D) gel electrophoresis and MALDI-TOF mass spectrometry, we compared and analyzed the cell lysate proteins of Mtb sequential clinical isolates from a patient undergoing anti-TB treatment. The mRNA expression levels of selected identified proteins were determined by quantitative real-time polymerase chain reaction (qRT-PCR). The genotypes of all four isolates remained homologous, indicating no re-infection. The initial isolate (before treatment) was sensitive to all first-line drugs, but the consecutive isolates were found to be resistant to isoniazid (INH) and rifampicin (RIF) and developed mutations in the katG, inhA and rpoB. the intensities of 27 protein spots were found to be consistently overexpressed in INH and RIF resistant isolates. The most prominent and overexpressed proteins found during the development of drug resistance were GarA (Rv1827), wag31 (Rv2145c), Rv1437 and Rv2970c. This preliminary proteomic study provides an insight about the proteins that are upregulated during drug resistance development. These upregulated proteins, identified here, could prove useful as immunodiagnostic and possibly drug resistant markers in future. However, more studies are required to confirm these findings.
    The Indian Journal of Medical Research 01/2015; 141(1):27-45. DOI:10.4103/0971-5916.154492
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    ABSTRACT: Background & objectives: Tuberculosis is a major health problem in India, and the emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) strains of Mycobacterium tuberculosis (Mtb) has further complicated the situation. Though several studies characterizing drug sensitive and drug resistant strains are available in literature, almost all studies are done on unrelated strains. Therefore, the objective of this study was to compare the proteomic data of four sequential isolates of M. tuberculosis from a single patient who developed MDR-TB during the course of anti-tuberculosis therapy (ATT). Methods: In this study, using two-dimensional (2D) gel electrophoresis and MALDI-TOF mass spectrometry, we compared and analyzed the cell lysate proteins of M. tuberculosis sequential clinical isolates from a patient undergoing anti-TB treatment. The mRNA expression levels of selected identified proteins were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Results: The genotypes of all four isolates remained homologous, indicating no re-infection. The initial isolate (before treatment) was sensitive to all first-line drugs, but the consecutive isolates were found to be resistant to isoniazid (INH) and rifampicin (RIF) and developed mutations in the katG, inhA and rpoB. The intensities of 27 protein spots were found to be consistently overexpressed in INH and RIF resistant isolates. The most prominent and overexpressed proteins found during the development of drug resistance were GarA (Rv1827), wag31 (Rv2145c), Rv1437 and Rv2970c. Interpretation & conclusions: This preliminary proteomic study provides an insight about the proteins that are upregulated during drug resistance development. These upregulated proteins, identified here, could prove useful as immunodiagnostic and possibly drug resistant markers in future. However, more studies are required to confirm these findings.
    The Indian Journal of Medical Research 01/2015; 141(1):27-45.
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    ABSTRACT: BACKGROUND & OBJECTIVES: Tuberculosis is a major health problem in India, and the emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) strains of Mycobacterium tuberculosis (Mtb) has further complicated the situation. Though several studies characterizing drug sensitive and drug resistant strains are available in literature, almost all studies are done on unrelated strains. Therefore, the objective of this study was to compare the proteomic data of four sequential isolates of Mtb from a single patient who developed MDR-TB during the course of anti-tuberculosis therapy (ATT). METHODS: In this study, using two-dimensional (2D) gel electrophoresis and MALDI-TOF mass spectrometry, we compared and analyzed the cell lysate proteins of Mtb sequential clinical isolates from a patient undergoing anti-TB treatment. The mRNA expression levels of selected identified proteins were determined by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: The genotypes of all four isolates remained homologous, indicating no re-infection. The initial isolate (before treatment) was sensitive to all first-line drugs, but the consecutive isolates were found to be resistant to isoniazid (INH) and rifampicin (RIF) and developed mutations in the katG, inhA and rpoB. the intensities of 27 protein spots were found to be consistently overexpressed in INH and RIF resistant isolates. The most prominent and overexpressed proteins found during the development of drug resistance were GarA (Rv1827), wag31 (Rv2145c), Rv1437 and Rv2970c. INTERPRETATION & CONCLUSIONS: This preliminary proteomic study provides an insight about the proteins that are upregulated during drug resistance development. These upregulated proteins, identified here, could prove useful as immunodiagnostic and possibly drug resistant markers in future. However, more studies are required to confirm these findings.
    The Indian Journal of Medical Research 01/2015; 141(1):27-45.
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    Amit Singh, Krishnamoorthy Gopinath, Niti Singh, Sarman Singh
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    ABSTRACT: Tuberculosis (TB) is caused by Mycobacterium tuberculosis (MTB) and the disease has remained a major health problem in most of the developing countries, particularly after the emergence of multidrug-resistant TB (MDR-TB). The MDR-TB is an intriguing subject and very little is known about the in vivo processes which take place during the acquisition of MDR. This study describes a unique case of pulmonary TB (PTB) from which four sequential isolates of MTB could be isolated while the patient was on anti-tubercular treatment. The first baseline isolate was sensitive to all drugs, but the subsequent three isolates acquired resistance to multiple drugs and finally the patient died after 27 months post-diagnosis when his fourth isolate became resistant to isoniazid, rifampicin, ethambutol and kanamycin. All sequential cultures were identified as MTB using conventional and molecular methods, including 16s RNA sequencing and the spoligotyping. Spoligotyping followed by comparison with SITVITWEB database revealed that all the isolates belonged to the family of the Central Asian Strain Delhi (CAS1_Delhi, ST26) genotype, and no cross or mixed infections were observed. The drug resistance was further characterized at the molecular level by sequencing the target genes (katG, inhA, rpoB, embB, eis promoter region and rrs). The results revealed mutated alleles associated with resistance to the respective drugs. This unique case indicates that it is possible to isolate MTB during treatment if the strain is acquiring resistance. The data presented from four sequential isolates provides an insight into what sequential genetic and proteomic changes occur in the bacteria during the in vivo acquisition of MDR.
    11/2013; 3(1). DOI:10.1016/j.ijmyco.2013.10.006
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    ABSTRACT: Tuberculosis is caused by Mycobacterium tuberculosis (MTB) and the disease has remained a major health problem in most of the developing countries, especially w here HIV is co-endemic. Recently, the disease has also re-emerged as the multi-drug resistant (MDR) and extensively drug resistant (XDR) forms of tuberculosis. To find effective drugs against these resistant forms of M. tuberculosis is a major challenge for researchers. How ever, the biggest hurdle to find such drugs is lack of know ledge about novel targets in the pathogen. It is mainly because the sequential isolates of M. tuberculosis from patients defaulting on anti-tuberculosis treatment have not been characterized for molecular entities, w hich may be responsible for resistance to anti-tubercular drugs. Such sequential isolates turning from sensitive to resistant form may provide better understanding in the discovery of biomarkers of drug resistance. We w ere successful in culturing 4 consecutive isolates, w hich transformed from sensitive to resistant forms over a period of 27 months. We have found that w hile the initial isolate w as sensitive to all four first line drugs, the sequential isolates became resistant to INH and RIF (MDR). 2D gel electrophoresis revealed that 27 protein spots w ere differentially expressed in resistant strains. We found that most prominent of these differentially regulated proteins w ere GarA, w ag31, Rv1437, Rv2970c. Our results suggest the possibility of using these proteins as immunodiagnostic and possible drug resistance markers.
    FEMS 2013, Leipzig, Germany; 07/2013
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    9th Sir Dorabji TATA Sympoium "Äntimicrobial Resistance", Banglore; 01/2013
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    ABSTRACT: Delayed or missed diagnosis of TB continues to fuel the global TB epidemic, especially in resource limited settings. Use of serology for the diagnosis of tuberculosis, commonly used in India, is another factor. In the present study a commercially available serodiagnostic assay was assessed for its diagnostic value in combination with smear, culture and clinical manifestations. A total of 2300 subjects were recruited for the study, but 1041 subjects were excluded for various reasons. Thus 1259 subjects were included in the study of which 470 were pulmonary tuberculosis cases (440 of 470 were culture-positive) and 789 were their asymptomatic contacts. A house-to-house survey method was used. Blood samples were tested for IgM, IgA, and IgG antibodies using the Pathozyme Myco M (IgM), Myco A (IgA) and Myco G (IgG) enzyme immunoassay (EIA). Out of 470 PTB cases, BCG scar was positive in 82.34%. The Mantoux test and smear positivity rates in PTB cases were 94.3% (430/456), and 65.32% (307/470), respectively. Among the asymptomatic contacts, BCG scar was positive in 95.3% and Mantoux test was positive in 80.66% (442/548) contacts. No contact was found falsely smear positive. The sensitivity of IgM, IgA, and IgG EIA tests was 48.7%, 25.7% and 24.4%, respectively, while the specificity was 71.5%, 80.5%, 76.6%, respectively. Performance of EIAs was not affected by the previous BCG vaccination. However, prior BCG vaccination was statistically significantly (p = 0.005) associated with Mantoux test positivity in PTB cases but not in contacts (p = 0.127). The agreement between serology and Mantoux test was not significant. The commercial serological test evaluated showed poor sensitivity and specificity and suggests no utility for detection of pulmonary tuberculosis.
    PLoS ONE 07/2012; 7(7):e40213. DOI:10.1371/journal.pone.0040213
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    Sarman Singh, Krishnamoorthy Gopinath
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    ABSTRACT: Mycobacterium avium subsp. paratuberculosis (MAP) is a well-established etiological agent of Johne's disease in animals. In humans, similar clinical condition, first described by Crohn as regional ileitis in 1932, now known as Crohn's diseases (CD), has also been associated with this mycobacterial species. However, there are two schools of thoughts, one favoring MAP as its etiological agent while the second considers it as an immune-inflammatory condition triggered by an external factor. Onset of CD requires a series of events including predisposition of certain inherited genetic traits, associated environmental stimuli, and immune-inflammatory response. A combination of these factors probably leads to this disease. Recently, some human genes have also been identified which regulate ability to respond appropriately to the external factors. Added to these factors are concerns about the selection of clinical specimens and poor adherence to laboratory quality controls. The literature is full of contradictory findings, but there a lack of uniformity in the materials and methods used by many of these researchers. In this review, we provide our perspective under above circumstances and give our point of view which may open a platform for debate regarding the MAP as the etiological agent of human CD.
    Journal of laboratory physicians 07/2011; 3(2):69-74. DOI:10.4103/0974-2727.86836
  • Sarman Singh, K Gopinath, Ashok Rattan
    Essential of Tuberculosis in Children., Edited by Vimlesh Seth, SK Kabra, 01/2011: chapter 6: pages 57-65; J P Brothers Medical Publishers Pvt. Ltd., New Delhi., ISBN: 978-93-5025-252-9
  • Sarman Singh, Krishnamoorthy Gopinath, Rachna Sood, Ashok Rattan
    IN, Essential of Tuberculosis in Children., 4 edited by Vimlesh Seth, S K Kabra, 01/2011: chapter 5: pages 41-56; J P Brothers Medical Publishers Pvt. Ltd., New Delhi.., ISBN: 978-93-5025-252-9
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    Krishnamoorthy Gopinath, Sarman Singh
    International Journal of Infectious Diseases 09/2010; 14(9):e826. DOI:10.1016/j.ijid.2010.07.002
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    Krishnamoorthy Gopinath, Sarman Singh
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    ABSTRACT: The infections due to non-tuberculous mycobacteria (NTM) are increasing worldwide, detrimentally affecting both HIV seronegative and immunocompromised individuals. Cases of non-tuberculous mycobacteria are reported mainly from European countries and America, where tuberculosis is not endemic. In TBendemic regions such as Southeast Asia and sub-Saharan Africa, the occurrence of NTM is under-reported. This article reviews the various possible hypotheses of reportedly low incidence and prevalence of NTM in TB-endemic countries, and the authors provide their own view point on this issue. The authors think that: 1. There is a lack of systemic reporting of non-tuberculous mycobacterial diseases due to overlapping clinical manifestations of tuberculosis and NTM diseases. 2. There is a lack of proper infrastructure for the identification of non-tuberculous mycobacteria in TB-endemic regions. 3. Because of the high burden of tuberculosis in these regions, the whole attention of health care workers and government is directed toward TB. 4. There is no geographical area or country unfit for the survival and spread of NTM. 5. The notion that some ethnic groups or races are inherently resistant to NTM infection has not been scientifically proven. 6. There is sufficient evidence that non-specific cross-immunity is developed due to latent tuberculosis against these less-virulent NTM. 7. There are no systemic regional surveys for evaluating the true prevalence of NTM. 8. Empirical use of fluoroquinolones and anti-tubercular drugs in relatively dysfunctional health care settings gives a false impression of a low incidence of non-tubercular cases.
    PLoS Neglected Tropical Diseases 04/2010; 4(4):e615. DOI:10.1371/journal.pntd.0000615
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    Krishnamoorthy Gopinath, Sarman Singh
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    ABSTRACT: Background: The diagnosis of pulmonary tuberculosis (PTB) is conventionally established by examination of three Ziehl—Neelsen stained smears; however, negative results do not preclude active TB. Since tubercle bacilli or their nucleic acids are also expected to be excreted through the kidneys, we assessed spot urine as a supplementary specimen for diagnosing PTB. Methods: A total of 164 respiratory specimens (147 sputum, 15 bronchoalveolar lavage, and two gastric lavage) from 81 suspected PTB cases were prospectively collected and processed. A total of 112 non-TB controls were also included in the study. For three consecutive days, morning urine specimens were collected from all patients and controls, and were processed for culture by BACTECTM MGIT 960 (mycobacteria growth indicator tube) and Lowenstein—Jensen methods and for PCR by amplifying a 441-bp fragment of the hsp65 gene (Mycobacterium genus-specific) and a 786-bp fragment of the cfp32 gene (TB complex-specific). Results: Of the 81 patients suspected of having PTB, 46 (56.8%) were sputum culture-positive. Of these, 12 (26.1%) were also urine culture-positive for Mycobacterium tuberculosis. Of the 35 sputum culture-negative cases, three (8.6%) were urine culture-positive. The TB complex specific PCR (cfp32) was positive in 52.2% (24/46) of the bacteriologically-confirmed and 28.6% (10/35) of the bacteriologically-negative PTB patients. In none of the control subjects were urine culture or PCR found to be positive for M. tuberculosis. Conclusions: Specific PCR and culture examination of spot urine samples from suspected PTB patients significantly improved the detection rate of PTB and should be encouraged in resource limited settings and where multiple pulmonary specimens are not feasible. #2008 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
    International journal of infectious diseases: IJID: official publication of the International Society for Infectious Diseases 12/2009; 13:374-379. DOI:10.1016/j.ijid.2009.11.001
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    Amit Singh, Krishnamoorthy Gopinath, Sarman Singh
    FEMS 2009, Gotenburg, Sweden; 06/2009
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    K Gopinath, S Singh
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    ABSTRACT: Polymerase chain reaction (PCR) is the most rapid and sensitive method for diagnosing mycobacterial infections and identifying the aetiological Mycobacterial species in order to administer the appropriate therapy and for better patient management. Two hundred and thirty-five samples from 145 clinically suspected cases of tuberculosis were processed for the detection of Mycobacterial infections by ZN (Ziehl Neelsen) smear examination, L-J & BACTEC MGIT-960 culture and multiplex PCR tests. The multiplex PCR comprised of genus-specific primers targeting hsp65 gene, Mycobacterium tuberculosis complex-specific primer targeting cfp10 (Rv3875, esxB) region and Mycobacterium avium complex-specific primer pairs targeting 16S-23S Internal Transcribed Spacer sequences. The multiplex PCR developed had an analytical sensitivity of 10 fg (3-4 cells) of mycobacterial DNA. The multiplex PCR test showed the highest (77.24%) detection rate, while ZN smear examination had the lowest (20%) detection rate, which was bettered by L-J culture (34.4%) and BACTEC MGIT-960 culture (50.34%) methods. The mean isolation time for M. tuberculosis was 19.03 days in L-J culture and 8.7 days in BACTEC MGIT-960 culture. Using the multiplex PCR, we could establish M. tuberculosis + M. avium co-infection in 1.3% HIV-negative and 2.9% HIV-positive patients. The multiplex PCR was also highly useful in diagnosing mycobacteraemia in 38.09% HIV-positive and 15.38% HIV-negative cases. The developed in-house multiplex PCR could identify and differentiate the M. tuberculosis and M. avium complexes from other Mycobacterial species directly from clinical specimens. The triplex PCR developed by us could be used to detect and differentiate M. tuberculosis, M. avium and other mycobacteria in a single reaction tube.
    Journal of Applied Microbiology 03/2009; 107(2):425-35. DOI:10.1111/j.1365-2672.2009.04218.x
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    Krishnamoorthy Gopinath, Sarman Singh
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    ABSTRACT: The diagnosis of pulmonary tuberculosis (PTB) is conventionally established by examination of three Ziehl-Neelsen stained smears; however, negative results do not preclude active TB. Since tubercle bacilli or their nucleic acids are also expected to be excreted through the kidneys, we assessed spot urine as a supplementary specimen for diagnosing PTB. A total of 164 respiratory specimens (147 sputum, 15 bronchoalveolar lavage, and two gastric lavage) from 81 suspected PTB cases were prospectively collected and processed. A total of 112 non-TB controls were also included in the study. For three consecutive days, morning urine specimens were collected from all patients and controls, and were processed for culture by BACTEC MGIT 960 (mycobacteria growth indicator tube) and Lowenstein-Jensen methods and for PCR by amplifying a 441-bp fragment of the hsp65 gene (Mycobacterium genus-specific) and a 786-bp fragment of the cfp32 gene (TB complex-specific). Of the 81 patients suspected of having PTB, 46 (56.8%) were sputum culture-positive. Of these, 12 (26.1%) were also urine culture-positive for Mycobacterium tuberculosis. Of the 35 sputum culture-negative cases, three (8.6%) were urine culture-positive. The TB complex specific PCR (cfp32) was positive in 52.2% (24/46) of the bacteriologically-confirmed and 28.6% (10/35) of the bacteriologically-negative PTB patients. In none of the control subjects were urine culture or PCR found to be positive for M. tuberculosis. Specific PCR and culture examination of spot urine samples from suspected PTB patients significantly improved the detection rate of PTB and should be encouraged in resource-limited settings and where multiple pulmonary specimens are not feasible.
    International journal of infectious diseases: IJID: official publication of the International Society for Infectious Diseases 12/2008; 13(3):374-9. DOI:10.1016/j.ijid.2008.07.022
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    Manimuthu Mani Sankar, Krishnamoorthy Gopinath, Roopak Singla, Sarman Singh
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    ABSTRACT: Non-tubercular mycobacteria (NTM) has not been given due attention till the recent epidemic of HIV. This has led to increasing interest of health care workers in their biology, epidemiology and drug resistance. However, timely detection and drug susceptibility profiling of NTM isolates are always difficult in resource poor settings like India. Furthermore, no standardized methodology or guidelines are available to reproduce the results with clinical concordance. To find an alternative and rapid method for performing the drug susceptibility assay in a resource limited settings like India, we intended to evaluate the utility of Tetrazolium microplate assay (TEMA) in comparison with proportion method for reporting the drug resistance in clinical isolates of NTM. A total of fifty-five NTM isolates were tested for susceptibility against Streptomycin, Rifampicin, Ethambutol, Ciprofloxacin, Ofloxacin, Azithromycin, and Clarithromycin by TEMA and the results were compared with agar proportion method (APM). Of the 55 isolates, 23 (41.8%) were sensitive to all the drugs and the remaining 32 (58.2%) were resistant to at least one drug. TEMA had very good concordance with APM except with minor discrepancies. Susceptibility results were obtained in the median of 5 to 9 days by TEMA. The NTM isolates were highly sensitive against Ofloxacin (98.18% sensitive) and Ciprofloxacin (90.09% sensitive). M. mucogenicum was sensitive only to Clarithromycin and resistant to all the other drugs tested. The concordance between TEMA and APM ranged between 96.4 - 100%. Tetrazolium Microplate Assay is a rapid and highly reproducible method. However, it must be performed only in tertiary level Mycobacteriology laboratories with proper bio-safety conditions.
    Annals of Clinical Microbiology and Antimicrobials 08/2008; 7:15. DOI:10.1186/1476-0711-7-15
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    K Gopinath, S Kumar, S Singh
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    ABSTRACT: The use of automated blood cultures system, such as MB/BacT, has provided a novel opportunity for laboratories to diagnose mycobacteremia in HIV-infected patients. However, no such study has been carried out in India so far. This prospective study was conducted on 52 HIV-positive patients with suspected tuberculosis who were referred to our tertiary care hospital in New Delhi. In these patients, the prevalence of mycobacteremia was evaluated using the MB/BacT automated culture system (bioMérieux, France). Twenty-seven HIV-negative but suspected tuberculosis patients were also included for comparison. Mycobacteria could be isolated from sputa or fecal samples of 20 HIV-positive patients (38.4%), and in nine (45%) of these 20 cases, mycobacteria could also be isolated simultaneously from their blood specimens. In the remaining 32 patients, all relevant non-hematological clinical samples remained negative for mycobacteria, but the pathogen could be detected from the blood samples of seven (21.87%) of these 32 patients. Therefore, only 25 (48%) clinically suspected patients remained negative in both Löwenstein-Jensen (L-J) and MB/BacT culture methods, and 12 of these responded to anti-tubercular treatment, while in the rest either non-tubercular diagnosis was established or they were lost to follow-up. The study revealed that low CD+(4) counts and poor or no reactivity to purified protein derivative (PPD) were the best clinical predictors for the occurrence of mycobacteremia in HIV-positive patients. Of the 16 isolates from blood, 13 were diagnosed as Mycobacterium tuberculosis and one each were identified as M. avium, M. kansasii, and a mixed infection of M. tuberculosis and M. avium complex. The prevalence rate of mycobacteremia was significantly low (11.1%) in HIV-negative patients. In conclusion, this study showed that blood culture could be an important adjunct investigation for confirming the clinical diagnosis of tuberculosis in HIV-positive patients.
    European Journal of Clinical Microbiology 07/2008; 27(6):423-31. DOI:10.1007/s10096-007-0450-x
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    Sarman Singh, Manimuthu Mani Sankar, Krishnamoorthy Gopinath
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    ABSTRACT: Fifty-four full-blown AIDS patients suspected of having HIV-tuberculosis co-infection were investigated for the prevalence of extensively drug-resistant (XDR) Mycobacterium tuberculosis. Out of the 54 patients, M. tuberculosis was isolated from 24 (44.4%). Twelve (50%) isolates of these had resistance to first-line drugs, whereas four (33.33%) were also resistant to second-line drugs. All four patients, in whom XDR M. tuberculosis was isolated, died within 2.6 months of diagnosis.
    AIDS 12/2007; 21(17):2345-7. DOI:10.1097/QAD.0b013e3282f125c9