Rapid diagnosis of extensively drug-resistant tuberculosis by use of a reverse line blot hybridization assay.

Drug resistance in tuberculosis (TB) is a matter of grave concern for TB control programs, as there is currently no cure for some extensively drug-resistant (XDR) strains. There is concern that this resistance could transmit, stressing the need for additional control measures, rapid diagnostic methods, and newer drugs for treatment. We developed an in-house assay that can rapidly detect resistance to drugs involved in the definition of XDR-TB directly from smear-positive specimens. Two hundred fifteen phenotypically XDR-TB isolates and 50 pansusceptible isolates were analyzed using a reverse line blot hybridization (RLBH) assay. The assay was also successfully applied to 73 smear-positive clinical specimens. The RLBH assay exhibited good sensitivity for the detection of resistance to isoniazid (99%), rifampin (99%), fluoroquinolones (95.3%), and second-line aminoglycosides (94.8%). The results from application of this assay on direct smear-positive clinical specimens revealed 93% concordance with the phenotypic drug susceptibility test (DST) results for the above-mentioned drugs. The time to accurate DST results was significantly reduced from weeks to 3 days. This molecular assay is a highly accurate tool for screening for XDR-TB, which achieves a substantial reduction in diagnostic delays.

Mutation detection and accurate diagnosis of extensively drug-resistant tuberculosis: report from a tertiary care center in India.

We screened and spoligotyped 150 consecutive phenotypically confirmed extensively drug-resistant Mycobacterium tuberculosis (XDR-TB) isolates (January 2008 to March 2009) for rifampin, isoniazid, fluoroquinolone, and aminoglycoside resistance targeting rpoB, inhA, katG, gyrA, gyrB, and rrs. Mutations predominant among XDR-TB were S315T (katG) (100% of isolates), S531L (rpoB) (97% of isolates), D94G (gyrA) (53% of isolates), and A1401G (rrs) (71% of isolates). Spoligotyping revealed 62% of the isolates to be Beijing.

Antimicrobial susceptibility profiles of meticillin-susceptible and -resistant Staphylococcus aureus: focus on daptomycin minimum inhibitory concentrations at a tertiary care centre in Mumbai, India.

In this study, daptomycin minimum inhibitory concentrations (MICs) for Staphylococcus aureus isolates were determined using Etest strips and were correlated with staphylococcal cassette chromosome mec (SCCmec) types and Panton-Valentine leukocidin (PVL) gene positivity. In total, 60 meticillin-resistant S. aureus (MRSA) and 60 meticillin-susceptible S. aureus (MSSA) isolates from clinical samples were subjected to antimicrobial susceptibility testing by disk diffusion according to Clinical and Laboratory Standards Institute guidelines, polymerase chain reaction (PCR) detection of PVL and mecA genes, and SCCmec typing. Daptomycin MICs were determined using Etest strips. The mecA gene was present in all MRSA isolates and was absent in 59 MSSA isolates. The PVL gene was present in 41 MRSA isolates and 25 MSSA isolates. Amongst the MRSA isolates, 10 were SCCmec type III, 27 were SCCmec IV and 23 were SCCmec V. Moreover, 26 SCCmec IV and 15 SCCmec V isolates were PVL-positive. All SCCmec III isolates were multidrug-resistant and PVL-negative. Daptomycin MICs ranged from 0.047 microg/mL to 1 microg/mL for MRSA and from 0.19 microg/mL to 1 microg/mL for MSSA. All MRSA and MSSA isolates were susceptible to daptomycin. Although SCCmec III MRSA and PVL-positive MSSA were resistant to more antimicrobial classes than SCCmec IV and V MRSA and PVL-negative MSSA, there does not appear to be a significant correlation between SCCmec types, PVL positivity and daptomycin MICs. MICs were not as low as expected for a newly introduced drug.

Molecular characterization of methicillin-resistant Staphylococcus aureus with emergence of epidemic clones of sequence type (ST) 22 and ST 772 in Mumbai, India.

A total of 412 methicillin-resistant Staphylococcus aureus (MRSA) strains isolated between October 2006 and June 2009, representing a mixed hospital- and community-associated patient population from Mumbai, India, were evaluated. MRSA was characterized by multiplex PCR amplification of the Panton-Valentine leukocidin (PVL) gene and the mecA gene, staphylococcal cassette chromosome mec (SCCmec) typing, and multilocus sequence typing (MLST). PCR results were compared with patient risk factors (CDC guidelines) and antimicrobial susceptibility profiles. A total of 395 MRSA strains were mecA positive, and 224 were PVL gene positive. A total of 97 mecA-positive strains were SCCmec III (25%), 136 were SCCmec IV (34%), and 162 were SCCmec V (41%). All SCCmec III strains were multidrug resistant, and all patients had risk factors. Of the SCCmec IV and V strains, 73% were multidrug susceptible and 72% of the associated patients had no risk factors. The multidrug susceptibility and absence of patient risk factors in 72% of cases with SCCmec IV and SCCmec V MRSA demonstrate the presence of community-associated MRSA (CA-MRSA) in Mumbai. Twenty-one percent of these patients had risk factors, signifying CA-MRSA infiltration into hospitals. MLST showed clonal expansion of multidrug-susceptible sequence type (ST) 22 (SCCmec IV) and ST 772 (SCCmec V), both of which feature in Asian studies and may be slowly replacing the multidrug-resistant ST 239 (SCCmec III) in hospitals. The PVL gene-positive methicillin-sensitive S. aureus (MSSA) strains were ST 30 and were postulated to be related to the penicillin-resistant S. aureus phage type 80/81, notorious for its virulence in the 1950s.

Comparison of phenotypic and genotypic methods for pyrazinamide susceptibility testing.

OBJECTIVES: To evaluate Pyrazinamide (PZA) susceptibility results obtained by phenotypic MGIT 960 TB system against enzymatic Pyrazinamidase assay and genotypic pncA gene sequencing. To find the prevalence of infections caused by M. bovis in PZA resistant M. tuberculosis complex isolates. METHODS: 33 consecutive PZA resistant and 30 consecutive PZA susceptible isolates reported for PZA susceptibility testing by MGIT 960 TB system were included in this study. Presence of active pyrazinamidase enzyme was sought by using the Wayne assay. The pncA gene was amplified by PCR and then sequenced to screen mutations. All the PZA resistant isolates were further spoligotyped to identify M. bovis, if present. RESULTS: Of 33 PZA resistant strains by MGIT 960, 31 were Wayne assay negative and two were positive. Of the 30 susceptible PZA strains six were Wayne assay negative reporting false resistance. PncA gene sequencing revealed that 32 of the 33 MGIT PZA resistant isolates had diverse nucleotide changes scattered throughout the pncA gene (one isolate did not show any mutation). Of the 30 phenotypically susceptible isolates, 21 were wild types whilst nine isolates showed the presence of a silent mutation C-T at codon 195. Fifteen mutations found in this study has not been described earlier. Not a single isolate of M. bovis was detected among PZA resistant M. tuberculosis complex isolates. CONCLUSION: MGIT 960 showed better concordance with sequencing results in comparison with Wayne assay. In present study, a high proportion (85%) of MDR-TB isolates from patients receiving anti-TB treatment were found to be resistant to PZA.

Osteoarticular tuberculosis--diagnostic solutions in a disease endemic region.

BACKGROUND: We conducted a study of osteoarticular tuberculosis in patients from private and public settings in a disease endemic area. Our objective was to assess the role of mycobacterial culture and polymerase chain reaction (PCR) in the diagnosis of osteoarticular tuberculosis (TB) in settings where only clinical and imaging diagnosis form the basis for treatment. METHODOLOGY: Ninety-three consecutive specimens collected from clinically suspected patients of osteoarticular TB were screened for bacterial culture, mycobacterial culture and in-house nested PCR. In addition, specimens were examined by imaging and histopathology. Ten specimens collected from patients suffering from other bone diseases were included as negative controls. RESULTS: Of the 93 clinically suspected TB patients, mycobacterial culture was positive for Mycobacterium tuberculosis (MTB) in 47 (51%) patients who were confirmed as definite TB cases. Of the remaining patients, 16 (17%) were diagnosed as probable, 19 (20%) as possible, and 11 (12%) as only clinically suspected TB cases. In-house nested PCR was positive in 65 (70%) cases. Fifteen patients were resistant to one or more anti-tuberculous drugs; twelve patients were multi-drug resistant, two of whom were extensively drug resistant. CONCLUSION: Mycobacterial cultures using liquid media with susceptibility should form the backbone of management of osteoarticular TB. Nested PCR enhances the sensitivity if performed in addition to culture.

Prospective comparison of eubacterial PCR and measurement of procalcitonin levels with blood culture for diagnosing septicemia in intensive care unit patients.

Rapid identification of infection has a major impact on the clinical course, management, and outcome of critically ill intensive care unit (ICU) patients. We compared the results of PCR and procalcitonin with blood culture for ICU patients suspected of having septicemia. Ninety patients (60 patients meeting the criteria for sepsis and 30 patients not meeting the criteria for sepsis) were evaluated. Compared with blood culture as the gold standard, the sensitivity, specificity, and positive and negative predictive values for PCR were 100%, 43.33%, 46.87%, and 100%, respectively, and for procalcitonin were 100%, 61.66%, 56.6%, and 100%, respectively. The average times required to produce a final result were as follows: PCR, 10 h; blood culture, 33 h; procalcitonin, 45 min. Both PCR and procalcitonin may be useful as rapid tests for detecting septicemia but compared with blood cultures lacked specificity.

Rapid speciation of 15 clinically relevant mycobacteria with simultaneous detection of resistance to rifampin, isoniazid, and streptomycin in Mycobacterium tuberculosis complex.

OBJECTIVE: To design and standardize an in-house reverse line blot hybridization (RLBH) assay for the accurate identification of 15 clinically relevant species of mycobacteria and for the detection of drug resistance to rifampin (RIF), isoniazid (INH), and streptomycin (STR) in Mycobacterium tuberculosis complex (MTB). MATERIAL AND METHODS: Oligonucleotides specific for 15 different species of mycobacteria and wild type and mutant alleles of selected codons in the rpobeta, inhA, katG, rpsL, and rrs genes were designed and immobilized on a membrane. A multiplex PCR was standardized to amplify all target genes. The assay was optimized using ATCC and known mutant strains. Three hundred MTB isolates, 85 non-tuberculous mycobacteria (NTM) isolates, and 48 smear-positive specimens were analyzed. Results were confirmed by PCR restriction enzyme assay and sequencing. RESULTS: Upon RLBH analysis, among the NTM, 14% were identified as Mycobacterium fortuitum, 16% were identified as Mycobacterium abscessus, 20% showed 99% homology with Mycobacterium intracellulare, and 31% showed 98% homology with Mycobacterium simiae. Of the 300 MTB isolates analyzed, 75% RIF-resistant isolates had Ser531Leu mutation in the rpobeta gene. Of the INH-resistant isolates, 89% showed Ser315Thr mutation in the katG gene, whereas 16% showed -15 C-->T mutation in the promoter region of the inhA gene. Among STR-resistant isolates, 75% had A-->G mutation in the rpsL gene at codon 43. RLBH results showed 96-99% concordance with phenotypic culture results. CONCLUSION: This is a first attempt at combining speciation with detection of drug resistance to RIF, INH, and STR in MTB for accurate and rapid management of mycobacterial infections as well as for compiling genotypic epidemiological data.

How good is compliance with surgical antibiotic prophylaxis guidelines in a tertiary care private hospital in India? A prospective study.

PURPOSE: There is a need to study compliance with surgical antibiotic prophylaxis guidelines in India. METHODS: In this prospective study, 100 consecutive surgical procedures performed at a tertiary care private hospital in Mumbai, India were observed. The choice of antibiotic, timing and duration of administration were recorded and compared to the hospital guidelines. RESULTS: Appropriateness of choice of antibiotic was seen in 68%, timing in 89%, dose in 75% and duration in 63% of cases. Hundred percent compliance to all criteria was observed in 52% of cases. The SSI rate was 3.3%. CONCLUSIONS: These compliance rates though suboptimal are similar to those reported in world literature. There is an urgent need to improve compliance with optimal surgical antibiotic prophylaxis guidelines so as to reduce risk of SSI and to prevent resistance and costs potentially associated with antibiotic misuse.

International Nosocomial Infection Control Consortium report, data summary for 2002-2007, issued January 2008.

We report the results of an International Nosocomial Infection Control Consortium (INICC) surveillance study from 2002 through 2007 in 98 intensive care units (ICUs) in Latin America, Asia, Africa, and Europe. During the 6-year study, using Centers for Disease Control and Prevention (CDC) National Nosocomial Infections Surveillance System (NNIS) definitions for device-associated health care-associated infection, we collected prospective data from 43,114 patients hospitalized in the Consortium's hospital ICUs for an aggregate of 272,279 days. Although device utilization in the INICC ICUs was remarkably similar to that reported from US ICUs in the CDC's National Healthcare Safety Network, rates of device-associated nosocomial infection were markedly higher in the ICUs of the INICC hospitals: the pooled rate of central line-associated bloodstream infections (CLABs) in the INICC ICUs, 9.2 per 1000 CL-days, is nearly 3-fold higher than the 2.4-5.3 per 1000 CL-days reported from comparable US ICUs, and the overall rate of ventilator-associated pneumonia was also far higher, 19.5 vs 1.1-3.6 per 1000 ventilator-days, as was the rate of catheter-associated urinary tract infection, 6.5 versus 3.4-5.2 per 1000 catheter-days. Most strikingly, the frequencies of resistance of Staphylococcus aureus isolates to methicillin (MRSA) (80.8% vs 48.1%), Enterobacter species to ceftriaxone (50.8% vs 17.8%), and Pseudomonas aeruginosa to fluoroquinolones (52.4% vs 29.1%) were also far higher in the Consortium's ICUs, and the crude unadjusted excess mortalities of device-related infections ranged from 14.3% (CLABs) to 27.5% (ventilator-associated pneumonia).

Broad-range bacterial and fungal DNA amplification on vitreous humor from suspected endophthalmitis patients.

BACKGROUND: Prompt and accurate diagnosis of infectious endophthalmitis is crucial for rapid and effective treatment. By identifying whether the causative pathogen is bacterial or fungal, a rational approach for the use of antibacterials or corticosteroids, respectively, can be followed. AIM: To assess the clinical utility of broad-range bacterial and fungal DNA amplification in the detection of endophthalmitis (postoperative, posttraumatic, and endogenous). METHODS: In a prospective study, vitreous humor samples from 70 patients with the clinical diagnosis of presumed endophthalmitis, and from 30 patients undergoing surgery for non-infectious causes, were subjected to routine microbiologic and molecular investigation. DNA extracted from a 50 microL sample was amplified by primers targeting the conserved 16S and 18S ribosomal RNA gene sequences of bacteria and fungi, respectively. Reagents for bacterial DNA amplification were decontaminated of endogenous DNA using 8-methoxypsoralen and long wave UV treatment. RESULTS AND DISCUSSION: A total of 35 specimens were positive for bacteria or fungi by culture. Of these, Gram-positive organisms were isolated in 19 specimens, Gram-negative organisms in 13 specimens and fungi in 3 specimens. Pseudomonas species, coagulase-negative Staphylococcus, and Streptococcus species were the main etiological agents isolated. Bacterial DNA amplification resulted in 49 positive specimens, compared with 32 positive specimens by culture; and fungal DNA amplification resulted in 11 positive specimens, compared with 3 positive specimens by culture. All control specimens were negative for both culture and DNA amplification. CONCLUSION: DNA extracted using a single-extraction protocol from 50 microL of vitreous humor and amplified with broad-range bacterial and fungal primers will enable the rapid differentiation (within 14 hours) between bacterial and fungal endophthalmitis and allow tailoring of therapy to individual patients.

An improved method of elimination of DNA from PCR reagents.

OBJECTIVE: The presence of exogenous DNA in commercially available polymerase chain reaction (PCR) reagent preparations is a serious problem when amplifying conserved regions of bacteria. The preferred and currently in-use method of decontamination using 8-methoxypsoralen (8-MOP) and UVA requires re-standardization of decontamination with increasing concentrations of 8-MOP and UVA irradiation timings, if the DNA load of reagents is high due to lot-to-lot differences. The objective of this study was to develop a decontamination method, which would (i) work at the minimum reported concentration of 8-MOP and UVA irridation timings; and (ii) take care of inter-batch DNA-load variability of reagents. MATERIALS AND METHODS: The improved method described here was formulated after studying the exact molecular mechanism of action of 8-MOP with DNA. The successful working of the method was experimentally proven and validated with 6-7 new batches of PCR reagents. The sensitivity of eubacterial PCR, after using the new method of decontamination, to be used clinically was checked with both the spiked specimens and the actual clinical specimens. RESULTS AND DISCUSSION: The new method was found to work at the same starting parameters of 8-MOP and UVA in such situations. The increased efficiency of this method was found to be due to the synergistic effect of both the selective treatment of Taq DNA polymerase and the split-irradiation approach.

High incidence of the Beijing genotype among multidrug-resistant isolates of Mycobacterium tuberculosis in a tertiary care center in Mumbai, India.

We report a high frequency (35%) of the Beijing genotype among multidrug-resistant isolates recovered in and around Mumbai, India. Further restriction fragment-length polymorphism typing showed that these strains were closely related. We also report a high frequency of the Delhi genotype (31% of isolates). Our data indicate considerable ongoing transmission of multidrug-resistant Mycobacterium tuberculosis strains of the Beijing genotype in Mumbai.

Comparing serology, antigenemia assay and polymerase chain reaction for the diagnosis of cytomegalovirus infection in renal transplant patients.

BACKGROUND: Cytomegalovirus (CMV) disease is responsible for significant morbidity and mortality following renal transplantation. Currently serology is the only method widely available in our country. Newer methods like early CMV pp65 antigenemia assay and CMV DNA amplification can diagnose CMV disease in its very early period. AIM: The aim of our study was to compare serologic method with antigenemia assay and CMV DNA amplification to diagnose CMV. METHODS: Seventy-three renal transplant recipients (from 7 centres) with clinical suspicion of CMV disease were studied prospectively. The diagnosis of CMV infection was suspected on the basis of fever and leucopenia. RESULT AND DISCUSSION: Three tests were done in all 73 patients and in 22 healthy subjects (control group). The sensitivity and specificity of serological test (CMV IgM) was 72.97 and 62.06%; of antigenemia assay was 89.18 and 100% and of PCR was 100 and 72.41%. CONCLUSION: Antigenemia assay is a sensitive and specific test for early and rapid diagnosis of CMV infection. Qualitative PCR is a sensitive marker but has low specificity.

Incidence of multidrug-resistant tuberculosis in urban and rural India and implications for prevention.

We compared the incidence of multidrug resistance in 150 consecutive Mycobacterium tuberculosis isolates obtained from a rural center (in Sakawar, India) and an urban tertiary care center (in Mumbai, India). The study highlights an alarmingly high percentage of multidrug-resistant M. tuberculosis isolates in Mumbai (51%) as compared with that at the rural center (2%).