Diagnosis of pleural tuberculosis by multi-targeted loop-mediated isothermal amplification assay based on SYBR Green I reaction: comparison with GeneXpert® MTB/RIF assay.

BACKGROUND: Diagnosis of pleural tuberculosis (TB) is tedious owing to its close resemblance with malignant pleural effusion and sparse bacterial load in clinical specimens. There is an immediate need to design a rapid and dependable diagnostic test to prevent unnecessary morbidity/mortality. RESEARCH DESIGN AND METHODS: A multi-targeted loop-mediated isothermal amplification (MT-LAMP) was deliberated using mpt64 and IS6110 to diagnose pleural TB within pleural fluids/biopsies. MT-LAMP products were analyzed by gel-based and visual detection methods, viz. SYBR Green I, SYBR Green I+deoxyuridine triphosphate uracil-N-glycosylase (dUTP-UNG), and dry methyl green reactions. RESULTS: In a pilot study, while assessing pleural TB/non-TB control subjects (n = 40), both SYBR Green I+dUTP-UNG/gel-based MT-LAMP assays exhibited better sensitivity/specificity than SYBR Green I and dry methyl green MT-LAMP. Since it is facile to work with SYBR Green I+dUTP-UNG than gel-based MT-LAMP, we validated the performance of SYBR Green I+dUTP-UNG in a higher number of specimens (n = 97), which revealed somewhat higher sensitivity (85.2 vs. 81.5%) and specificity (97.7 vs. 90.7%) than SYBR Green I MT-LAMP. Furthermore, the sensitivity attained by SYBR Green I+dUTP-UNG MT-LAMP was significantly higher (p < 0.001) than GeneXpert. CONCLUSIONS: Our SYBR Green I+dUTP-UNG MT-LAMP is a simple and reliable method to diagnose pleural TB, which may translate into a point-of-care test.

Identification of mycobacterial MPT-64 and ESAT-6 proteins in urogenital tuberculosis patients by real-time immuno-PCR.

Aim: Diagnosis of urogenital tuberculosis (UGTB) is difficult and there is an immediate need to develop a reliable diagnostic test. Methods: A real-time immuno-PCR (RT-I-PCR) was developed to identify a cocktail of MPT-64 + ESAT-6 in both male/female UGTB patients comprising five confirmed cases, 40 clinically suspected cases and 37 non-TB controls, from whom mid-stream urine specimens were collected, while endometrial biopsies of female patients were obtained on day 1 of their menstrual cycle. Results obtained by RT-I-PCR were compared with I-PCR/ELISA and GeneXpert. Results: A wide range (500 fg/ml-10 ng/ml) of MPT-64 + ESAT-6 was detected in UGTB specimens by RT-I-PCR, although ELISA showed a narrow range (2.5-11 ng/ml). Sensitivities of 80% and 82.2% were obtained by RT-I-PCR in clinically suspected and total UGTB cases, respectively, whereas 94.6% specificity was obtained. Concurrently, RT-I-PCR revealed significantly higher (p < 0.05-0.001) sensitivity than I-PCR/ELISA and GeneXpert. Conclusion: After improving the specificity, the authors may develop RT-I-PCR into a diagnostic kit.

Urogenital tuberculosis (UGTB) involves infection of the urinary tract and genital organs of male/female patients by Mycobacterium tuberculosis bacteria. Delayed diagnosis and therapy of UGTB lead to infertility and kidney failure. The routine tests used to detect the bacteria are not very sensitive due to low levels of bacteria present in UGTB specimens. Moreover, most nucleic acid amplification tests, such as PCR tests, give false-positive and false-negative results. The authors designed a real-time immuno-PCR test for detecting a cocktail of M. tuberculosis proteins in UGTB patients that revealed quite promising results, which were superior to immuno-PCR/ELISA and GeneXpert tests. After further improvement in the specificity and reduction of the price, this real-time immuno-PCR test could be used in routine diagnosis.

Comparative Evaluation of Serum High-sensitivity C-Reactive Protein and Complete Hemogram Indices in Subjects with and without Apical Periodontitis: A Prospective Interventional Study.

INTRODUCTION: This study aimed to compare the levels of high-sensitivity C-reactive protein (hsCRP) and complete hemogram (CH) parameters before and after root canal treatment in patients with apical periodontitis (AP) and healthy controls. METHODS: Twenty-five patients with asymptomatic AP in a single permanent tooth were recruited along with age- and sex-matched healthy controls. Baseline serum hsCRP and CH parameters were recorded in both groups. Root canal treatment was performed in teeth with AP, and biochemical parameters were re-evaluated at the 6-month follow-up. Mann-Whitney and chi-square tests were used to analyze data quantitatively and qualitatively, respectively. Spearman correlation was applied to explore the relation between hsCRP with AP and periapical healing. Multivariate linear regression tests were used to assess the effect of independent variables such as age, sex, body mass index, and periapical index score on levels of hsCRP. RESULTS: A baseline comparison between patients with AP (3.37 ± 2.69 mg/L) and controls (1.69 ± 2.2 mg/L) revealed a significant difference in hsCRP levels. However, all CH parameters were within the reference range. A total of 22 patients in the AP group completed follow-up, and based on the periapical index score and clinical presentations, 72.2% of patients were classified as healed. At follow-up, hsCRP significantly reduced to 1.79 ± 1.65 mg/L in the AP group. A significant correlation between AP and hsCRP was observed. CONCLUSIONS: Patients with AP had a significantly higher inflammatory burden than healthy controls, which significantly reduced after root canal treatment. No significant change was detected in CH indices.

Diagnosis of osteoarticular tuberculosis: multi-targeted loop-mediated isothermal amplification assay versus multiplex PCR.

Aim: Diagnosis of osteoarticular tuberculosis (OATB) is quite challenging and there is an urgent need to design a prompt and precise diagnostic test. Methods: We developed a multi-targeted loop-mediated isothermal amplification (LAMP) assay using mpt64 (Rv1980c) and pstS1 (Rv0934) targets for the detection of Mycobacterium tuberculosis in OATB patients. Results: The sensitivities of 100 and 82.4% were obtained in confirmed (n = 10) and suspected (n = 57) OATB cases, respectively by multi-targeted LAMP with a specificity of 96.9% (n = 33). Moreover, the sensitivities attained by multi-targeted LAMP in total OATB cases were significantly higher (p < 0.05-0.01) than multiplex PCR (mpt64 + pstS1) and GeneXpert assay. Conclusion: Our LAMP is simple, reliable and cost-effective method, which may develop into an attractive diagnostic kit for early detection of OATB cases.

Lay abstract Diagnosis of osteoarticular tuberculosis (OATB) or bone and joint TB caused by Mycobacterium tuberculosis (Mtb) is quite difficult owing to the low bacterial load present in OATB specimens, and difficulty of obtaining specimens since Mtb bacilli are only present deep inside the tissues. Mostly, diagnosis of OATB relies on clinical findings and imaging, which often mimic other pus-producing microbial infections and inflammatory arthritis, while the conventional bacteriological tests (smear/culture) almost fail. Therefore, we developed a multi-targeted loop-mediated isothermal amplification (LAMP) assay for early detection of OATB cases, which showed superiority over multiplex PCR and GeneXpert assay. Overall, our LAMP is straightforward, accurate and low-cost assay that may lead to the development of a diagnostic kit for routine use.

Diagnosis of tuberculosis by nanoparticle-based immuno-PCR assay based on mycobacterial MPT64 and CFP-10 detection.

Aim: To improve the diagnostic accuracy of immuno-PCR (I-PCR) in tuberculosis (TB) patients by using functionalized gold nanoparticles (AuNPs) coupled with detection antibodies and oligonucleotides, and magnetic beads (MBs) conjugated with capture antibodies in the liquid phase. Materials & methods: MB-coupled AuNP-based I-PCR (MB-AuNP-I-PCR) assay was designed to detect a cocktail of Mycobacterium tuberculosis MPT64 and CFP-10 proteins in bodily fluids of TB patients. Results: The sensitivities of 89.3 (n = 94) and 78.1% (n = 73) were observed in pulmonary TB and extrapulmonary TB patients, respectively, with specificities of 97.9-98.3%. Notably, the sensitivities attained by MB-AuNP-I-PCR in smear-negative pulmonary TB and extrapulmonary TB patients were significantly higher (p < 0.05-0.001) than Magneto-ELISA and GeneXpert assay. Conclusion: The improved technology, as well as enhanced diagnostic accuracy of MB-AuNP-I-PCR, may lead to development of an attractive diagnostic kit.

Quantitative detection of a cocktail of mycobacterial MPT64 and PstS1 in tuberculosis patients by real-time immuno-PCR.

AIM: There is an urgent need to design a reliable diagnostic test for tuberculosis (TB). METHODS: Real-time immuno-PCR (RT-I-PCR) assay was devised for the quantitative detection of a cocktail of mycobacterial MPT64 (Rv1980c) and PstS1 (Rv0934) in TB patients. RESULTS: A broad dynamic range of 0.95 pg/ml-95 ng/ml of MPT64+PstS1 was detected in TB patients. In smear-positive (n = 59) and smear-negative (n = 42) pulmonary TB cases, sensitivities of 93.2 and 83.3% were observed, respectively with 92.8% specificity, whereas a sensitivity of 77.9% and a specificity of 91.3% were observed in extrapulmonary TB cases (n = 86). Furthermore, significantly reduced MPT64+PstS1 concentrations (p < 0.001) were noticed in patients on therapy by RT-I-PCR as compared with untreated patients. CONCLUSION: Our RT-I-PCR assay revealed high sensitivity especially for the rapid diagnosis of smear-negative pulmonary TB and paucibacillary extrapulmonary TB samples, which could also monitor the dynamics of disease in patients on therapy.

Comparative evaluation of GeneXpert MTB/RIF and multiplex PCR targeting mpb64 and IS6110 for the diagnosis of pleural TB.

AIM: Diagnosis of pleural TB poses serious challenges due to paucibacillary nature of specimens and there is an urgent need to devise a reliable diagnostic test. METHODS: We compared GeneXpert Mycobacterium tuberculosis/rifampin assay and the multiplex PCR (M-PCR) targeting mpb64 (Rv1980c) and IS6110 in pleural fluids (n = 78) of pleural TB patients and non-TB controls. RESULTS: The sensitivities of 89.6 and 33.3%, and specificities of 96.7 and 100%, were observed with M-PCR and Xpert assay, respectively. CONCLUSION: M-PCR showed superiority over Xpert assay and may facilitate an efficient diagnosis of pleural TB.

Effect of Subgingivally Delivered 10% Emblica officinalis Gel as an Adjunct to Scaling and Root Planing in the Treatment of Chronic Periodontitis - A Randomized Placebo-controlled Clinical Trial.

Emblica officinalis fruit possesses varied medicinal properties including cytoprotective antimicrobial, antioxidant, antiresorptive and antiinflammatory activity. The present study aimed to investigate the effect of subgingival application of indigenously prepared E. officinalis (Amla) sustained-release gel adjunctive to scaling and root planing (SRP) on chronic periodontitis. Forty-six patients (528 sites) were randomly assigned to control group (23;264): SRP +placebo gel and test group (23;264): SRP + 10% E. officinalis gel application. Periodontal parameters: plaque index, gingival index, probing pocket depth (PPD), clinical attachment level (CAL) and modified sulcus bleeding index (mSBI) were assessed at baseline, 2 and 3-month post-therapy. Forty patients (470 sites) completed the trial. When test and control sites were compared, significantly more reduction in mean PPD, mSBI, number of sites with PPD = 5-6 mm, PPD ≥ 7 mm, CAL ≥ 6 mm and greater CAL gain were achieved in test sites at 2- and 3-month post-therapy (p < 0.05). Locally delivered 10% E. officinalis sustained-release gel used as an adjunct to SRP may be more effective in reducing inflammation and periodontal destruction in patients with chronic periodontitis when compared with SRP alone. Copyright © 2016 John Wiley & Sons, Ltd.

Comparative Evaluation of Several Gene Targets for Designing a Multiplex-PCR for an Early Diagnosis of Extrapulmonary Tuberculosis.

PURPOSE: Diagnosis of extrapulmonary tuberculosis (EPTB) poses serious challenges. A careful selection of appropriate gene targets is essential for designing a multiplex-polymerase chain reaction (M-PCR) assay. MATERIALS AND METHODS: We compared several gene targets of Mycobacterium tuberculosis, including IS6110, devR, and genes encoding MPB-64 (mpb64), 38kDa (pstS1), 65kDa (hsp65), 30kDa (fbpB), ESAT-6 (esat6), and CFP-10 (cfp10) proteins, using PCR assays on 105 EPTB specimens. From these data, we chose the two best gene targets to design an M-PCR. RESULTS: Among all gene targets tested, mpb64 showed the highest sensitivity (84% in confirmed cases and 77.5% in clinically suspected cases), followed by IS6110, hsp65, 38kDa, 30kDa, esat6, cfp10, and devR. We used mpb64+IS6110 for designing an M-PCR assay. Our M-PCR assay demonstrated a high sensitivity of 96% in confirmed EPTB cases and 88.75% in clinically suspected EPTB cases with a high specificity of 100%, taking clinical diagnosis as the gold standard. CONCLUSION: These M-PCR results along with the clinical findings may facilitate an early diagnosis of EPTB patients and clinical management of disease.

Acinetobacter lwoffii an emerging pathogen in neonatal ICU.

BACKGROUND: Acinetobacter species are ubiquitous in the environment and are important causative agent for nososcomial infection especially in immunocompromised patients. Multi drug resistant Acinetobacter lwoffii are emerging as a pathogen in neoanatal sepsis. AIMS AND OBJECTIVE: This study was aimed to evaluate the clinical and antibiotic profile of Acinetobacter lwoffii. MATERIAL AND METHODS: This study was done on blood samples from neonates admitted to neonatal intensive care unit during a period of one year from January to December 2012, who developed Acinetobacter infection. The diagnosis of isolates and antibiotic susceptibility testing was done by both conventional as well as by automated system. RESULTS: Out of total 13,133 blood samples received for culture, 1418(10.8%) were from NICU. Ninety (6.3%) isolates were found to be positive for the growth of Acinetobacter species. Of these isolates 31.11% were found to be Acinetobacter lwoffii, 68.9% were Acinetobacter baumannii calcaetius complex. Acinetobacter lwoffii isolates were most commonly sensitive to imepenem 16(57%), cotrimoxazole 9(32%), ciprofloxacin 6(21%) followed by amoxyclavulanic acid 2(7%) and cefuroxime 1(3.5%). CONCLUSION: Multi drug resistant Acinetobacter lwoffii infection is increasing particularly in premature and very low-birth weight neonates. Judicious and timely antibiotic use in NICUs are one of the important key in controlling multi-drug resistant Acinetobacter infection and improving clinical outcome.

Comparison of ELISA and Microscopy for detection of Cryptosporidium in stool.

BACKGROUND: Cryptosporidiosis, a diarrheal disease caused by the protozoan parasite Cryptosporidium spp. has become recognized as one of the most common causes of water borne diseases in humans. AIMS AND OBJECTIVES: To compare the sensitivity of ELISA and Microscopy for detection of Cryptosporidium in stool samples Materials and Methods: The study was conducted in the Department of Microbiology of PT. B.D. Sharma PGIMS Rohtak, between January 2011 to june 2011 on 50 stool samples, which were processed for detection of cryptosporidial antigen by ELISA and detection of cysts by microscopy (Modified Ziehl and Nelsen staining). STUDY AND DESIGN: This was a prospective study conducted in the Department of Microbiology in PT. BD Sharma, PGIMS, Rohtak, India. RESULT: Out of total, 50 stool samples eighteen (36%) samples were found positive for Cryptosporidium cysts by microscopy in comparison to 3(6%) stool samples which were found positive for cryptosporidial antigen by ELISA. Samples found positive with ELISA were also positive with microscopy. Sensitivity, specificity, positive predictive value and negative predictive value for ELISA was 16.7%, 100%, 100% and 68% respectively. CONCLUSION: The study concludes that stool microscopic Modified acid fast staining is more sensitive method than ELISA for detection of Cryptosporidium in stool samples but the specificity of ELISA was more than microscopy.

Dental caries, gingivitis, periodontitis: a review

Microbial populations colonizing the teeth and periodontal tissues are a major source of pathogens responsible for oral and dental infections including dental caries, gingivitis, periodontitis etc. Dental caries is a multifactor and infectious disease resulting dueto interaction of three different aspects like dietary sugar, susceptible tooth enamel and oral microbial colonization. Plaques from caries active sites have significantly higher proportion of Streptococcus mutans (principle acid producer) with pH levels of 5.0 or lower. Dental decay occurs when normal demineralization remineralization is disturbed. On the other hand the most common form of gingivitis is chronic or long standing plaque induced gingivitis while acute necrotizing ulcerative gingivitis is most aggressive, developing gingivitis is associated with increasing numbers of Actinomyces israeliwhereas gingivitis with bleeding is associated with A. viscosus and pigmented Bacteroides. Periodontitis is defined as loss of alveolar support to the tooth and can be differentiated microbiologically and clinically into adult, localised juvenile and pre-pubertal periodontitis. Various species of Bacteroides, Actinomyces, Fusobacterium etc. have been isolated from cases of active periodontitis. Thus wherever possible both aerobic and anaerobic culture should be performed and appropriate antibiotic therapy should be prescribed instead of empirical treatment.

Citrobacter bacteremia in a tertiary care hospital.

From January 1998 to December 2001, 176 cases of Citrobacter bacteremia occurred of which repeat isolation was possible in 48 cases. Of 48 isolates, 79.1% were C. diversus and 20.9% C. freundii. Citrobacter bacteremia was polymicrobial in 46.1% cases, and maximum number of cases (54.1%) occurred in the age group less than 10 years. Portal of entry was unknown (42.3%), respiratory tract (20.9%), gastrointestinal tract (15.3%) and urinary tract 15.3%. C. freundii isolates were relatively more resistant than C. diversus against 10 tested antimicrobial agents, while 79.1% isolates were multiresistant. Sensitivity based on MIC was highest for ceftizoxime, ciprofloxacin and cefotaxime. Overall mortality of Citrobacter bacteremia was seen in 56% of cases. Therefore greater caution is required in selection of antibiotic therapy in order to avoid selection of strains and treatment failure.