Diagnostic validation of rapid molecular detection of Mycobacterium tuberculosis in pus samples by GeneXpert.

OBJECTIVE: To evaluate the performance of GeneXpert for detection of mycobacterium tuberculosis in pus samples and compare its results with conventional techniques in terms of validity, rapidity and rifampicin resistance. METHODS: This longitudinal, descriptive study was conducted at Jinnah Hospital, Lahore, Pakistan, from January 2012 to December 2015, and comprised pus samples of people suspected of having extra-pulmonary tuberculosis. Participants were included by using consecutive sampling technique. The pus samples were subjected to Ziehl-Neelsen smear microscopy and Lowenstein-Jensen culture as per World Health Organisation's protocol and GeneXpert as per manufacturer protocol. SPSS 17 was used for data analysis. Validity of GeneXpert and rifampicin resistance were determined and compared with Ziehl-Neelsen staining using Lowenstein-Jensen culture as the gold standard. RESULTS: Of the 212 pus samples, 84(39.6%) were positive on Lowenstein-Jensen culture with mean turnaround time of 20±6 days, 77(36.3%) on GeneXpert and 22(10.4%) on Ziehl-Neelsen smear. The highest detection rate of mycobacterium tuberculosis 62(80.5%) was in lymph node samples by GeneXpert. The sensitivity and specificity of GeneXpert were 91.6% and 100% respectively, while Ziehl-Neelsen smear showed a sensitivity26.2% and specificity of 100%. Rifampicin resistance was detected in 5(6.4%) pus samples by GeneXpert. CONCLUSIONS: GeneXpert had a higher validity compared to Ziehl-Neelsen smear microscopy.

Acinetobacter Spp: Resistance and therapeutic decisions at the turn of the novel millennium.

This study was planned to evaluate sample wise isolation and antimicrobial resistant trends of Acinetobacter spp in different departments of a tertiary care hospital. This was a transversal descriptive study, carried out in the clinical microbiology laboratory of the Allama Iqbal Medical College/ Jinnah Hospital, Lahore, Pakistan, during the period of January 2015 to December 2016. Every clinical specimen was processed for bacterial culture and antimicrobial susceptibly testing. A total of 3590 (2015=1780, 2016=1810) clinical specimens were processed. Of the total, only 54.7% were gram-negative, among these Acinetobacter spp were isolated from 10.1% and 16.5% samples respectively in 2015-16 with an overall rate of 24.3%. The highest occurrence of Acinetobacter spp isolates was reported from Intensive care units (ICU) (54%) followed by surgical units (25%) and medical units (16%). It is noteworthy that ICU and internal medicine showed the highest resistance rates, whereas, lower resistance rate was observed for the outdoor patients (OPD). Although collistin showed 0% resistant while ceftriaxone, ciprofloxacin, gentamicin, and tigecycline showed 90%, 68%, 66%, 66% and 62% resistance against Acinetobacter spp. respectively. An alarming increase in the resistance rate of meropenem, cefoperazone/sulbactam, piperacillin/ tazobactam, ciprofloxacin, and imipenem was observed from the year 2015 to 2016. This startling resistance acquired by Acinetobacter spp. within a period of one year, represent very limited therapeutic options left for the infections caused by Acinetobacter spp. Unavailability of effective drugs and limited therapeutic options enforce the health care practitioners to prescribe expensive and broad range antibiotics, which may cause harm to the patient. Therefore, it is need of an hour to better understand the antimicrobial patterns and optimize antimicrobial prescription policies for the control of multidrug-resistant Acinetobacter spp.

Diagnosis of paediatric sepsis by automated blood culture system and conventional blood culture.

OBJECTIVE: To evaluate the time required for isolation of aerobic bacterial pathogen from paediatric septicaemia suspects by using BACTEC 9240 blood culture system, and to compare the results with conventional blood culture technique. METHODS: This comparative cross-sectional study was conducted at the Jinnah Hospital, Lahore, Pakistan, from July to December 2013, and comprised blood samples of suspected septicaemia children. The blood samples were inoculated into automated BACTEC 9240 Peds Plus/F resin-based media. At the same time, conventional blood culture bottle was also inoculated for comparison. The time of culture positivity and bacterial spectrum isolated from these samples was evaluated. SPSS 20 was used for data analysis. RESULTS: Of the 100 blood culture samples, 36(36%) were true pathogens on BACTEC 9240, while 24(24%) were positive through the conventional method. The mean age of the participants was 0.65±20 days. Staphylococcus aureus was detected in 12(12%) samples by BACTEC 9240 and in 7(7%) cases by conventional system. BACTEC 9240 significantly reduced time of positivity from 48 hours to 21 hours compared to conventional system. The number of samples detected within 36 and 48 hours was 7(19.4%) by BACTEC 9240 and 17(70.8%) by conventional system (p<0.05). CONCLUSIONS: Diagnosis of paediatric septicaemia through BACTEC 9240 was quicker with high yield and great sensitivity compared to the conventional technique.

GeneXpert: A new tool for the rapid detection of rifampicin resistance in mycobacterium tuberculosis.

OBJECTIVE: To evaluate the diagnostic accuracy of GeneXpert assay for the detection of rifampicin resistance in mycobacterium tuberculosis using conventional drug susceptibility testing as gold standard. METHODS: This cross-sectional study was conducted at Jinnah Hospital, Lahore, / Allama Iqbal Medical College, Lahore, Pakistan, from January 2012 to December 2014, and comprised clinically and radiologically diagnosed tuberculosis suspected cases. Pulmonary and extra-pulmonary specimens were collected from strong tuberculosis suspects. All specimens were processed for Ziehl Neelsen staining, Lowenstein-Jensen culture and GeneXpert assay. All mycobacterium tuberculosis positive cases on Lowenstein-Jensen culture were further processed for drug susceptibility testing. RESULTS: Of the 2,200 cases, 840(49.46%) were positive for mycobacterium tuberculosis on GeneXpert assay. Of them, 134(15.6%) cases showed rifampicin resistance on GeneXpert assay. The sensitivity, specificity, positive predictive value and negative predictive value of GeneXpert assay for rifampicin resistance were 127(98.3%), 704(99.1%), 127(94.7%) and 704(99.4%), respectively, by comparing the results with drug susceptibility testing. CONCLUSIONS: GeneXpert assay was an extremely helpful diagnostic tool for the detection of rifampicin resistance in tuberculosis suspects with fairly high sensitivity and specificity along with short turnout time.

Identification of TBc: Using MTP 64 protein and cord formation.

Tuberculosis (TB) is an airborne infectious disease caused by Mycobacterium tuberculosis. The genus Mycobacterium comprises over 150 species. Non-tuberculosis Mycobacteria are the cause of opportunistic infections and frequently presents with similar clinical features like tuberculosis so species identification is important for management. The current study was designed for presumptive diagnosis of MTB by detecting MPT 64 and Cord formation and the sensitivity and specificity of Cord Formation in MGIT positive samples. A cross sectional study consists of 100 MGIT positive samples. Ziehl-Neelsen Staining was performed to detect Cord Factor and TBc ID was undertaken to detect MPT 64 protein. Out of 100 MGIT positive samples 92 were positive for Cord factor and 08 were negative, whereas 89 samples were positive on MGIT TBc Identification Device and 11 were negative. The sensitivity for TBc ID was 94.6% and specificity was 100% with Positive Predictive Value and Negative Predictive Value of 98.9% and 81.8% respectively. TBc ID and Cord Factor seem to be highly sensitive and specific for rapid diagnosis and accurate differentiation Mycobacterium tuberculosis from Non-tuberculosis.

Silent killers: Transfusion Transmissible Infections-TTI, among asymptomatic population of Pakistan.

OBJECTIVE: To analyse transfusion transmissible infections in asymptomatic population. METHODS: This study was conducted at the Allama Iqbal Medical College and Jinnah Hospital, Lahore, Pakistan, from December 2014 to November 2015, and comprised healthy asymptomatic blood donors.Every sample was screened for the presence of antibodies/antigens of hepatitis C virus, human immunodeficiency virus, treponemapallidum, hepatitis B virus and malaria parasite through rapid immunochromatographic technique. RESULTS: Of the 18,274 blood donors, 17,276(94.53%) were found healthy and 998(5.46%) were infected. Besides, 71(0.38%) had multiple infections. The overall frequency of anti-hepatitis C virus, treponemapallidum (syphilis), hepatitis B surface antigen, malaria parasite and anti-human immunodeficiency virus was 480(2.62%), 284(1.55%), 210(1.10%), 20(0.10%) and 4(0.02%), respectively. CONCLUSIONS: Blood transfusion was found to be a significant but preventable mode of spread of transfusion transmissible infections.

Congenital Hypothyroidism in Neonates of a Tertiary Care Hospital.

OBJECTIVE: To determine neonatal congenital hypothyroidism among neonates born in a tertiary care hospital of Lahore Pakistan. METHODS: This cross-sectional study was carried out at Pathology Department of Allama Iqbal Medical College, Lahore in collaboration with Pediatrics and Gynecology & Obstetrics Department, Jinnah Hospital, Lahore Pakistan. A total of 770 babies were included in this study, both male and female. About 2 ml venous blood samples were collected aseptically from the neonates in sterile clotted tube. Serum was separated and serum TSH was determined by ELISA method. RESULTS: Out of total 770 neonates, 48.9% were female and 51.0% were males with the ratio of 1:1.04. Neonatal congenital hypothyroidisim (TSH, >30 mIU/L), was observed in 0.4% (Frequency, 1:257) nenates, with the incidence rate of 1:257. Female to male ratio of hypothyroid neonates was 2:1. The mode of delivery vise distribution showed, among n=251 neonates born by normal delivery, only a single case of hypothyroidism was detected, and among n=519 neonates delivered by cesarean section, only two neonates were belong to hypothyroidism. CONCLUSION: The frequency of Congenital Hypothyroidism is notably higher in pediatric community than reported in most other countries. This result emphasizes the necessity of a nationwide screening program.

N-acetylcysteine and azithromycin affect the innate immune response in cystic fibrosis bronchial epithelial cells in vitro.

BACKGROUND AND OBJECTIVE: We have previously reported that N-acetylcysteine (NAC), ambroxol and azithromycin (AZM) (partially) correct the chloride efflux dysfunction in cystic fibrosis bronchial epithelial (CFBE) cells with the ΔF508 homozygous mutation in vitro. METHODS: In the present paper, we further investigated possible immunomodulatory effects of these drugs on the regulation of the innate immune system by studying the expression of the cytosolic NOD-like receptors NLRC1 and NLRC2, and interleukin (IL)-6 production in CFBE cells. RESULTS: Under basal conditions, PCR and Western Blot data indicate that the NLRC2 receptor has a reduced expression in CF cells as compared to non-CF (16HBE) cells, but that the NLRC1 expression is the same in both cell lines. AZM significantly upregulated NLRC1 and NLRC2 while NAC upregulated only NLRC2 receptor expression in CF cells. Reduced basal IL-6 production was found in CF cells as compared to non-CF cells. MDP (an NLRC2 agonist), NAC and AZM, but not Tri-DAP (an NLRC1 agonist), increased IL-6 production in CF cells, indicating that in CF cells IL-6 upregulation is independent of NLRC1, but involves the activation of NLRC2. CONCLUSION: Overall, the results indicate that NAC and AZM not only can correct the chloride efflux dysfunction but also have a weakly strengthening effect on the innate immune system.

Rapid diagnosis of tuberculosis using Xpert MTB/RIF assay - Report from a developing country.

OBJECTIVE: To evaluate the diagnostic accuracy of the Xpert MTB/RIF assay for the detection of M. tuberculosis in pulmonary and extrapulmonary specimens and to compare it with conventional techniques. METHODS: During a period of 10 months from December 2012 through September 2013, two hundred and forty five clinically TB suspects were enrolled for Xpert MTB\RIF assay. The cohort comprised of 205 suspects of pulmonary TB and 40 of extrapulmonary TB (EPTB). The 40 EPTB samples included pus aspirated from different sites of the body (n=19), pleural fluid (n=11), ascitic fluid (n=7), pericardial fluid, CSF and urine one each. Ziehl-Neelsen (ZN) Stained smear microscopy, culture on LJ media and Xpert MTB/RIF assay was performed on samples from these patients. RESULTS: M. tuberculosis (MTB) were detected by Xpert MTB/RIF test in 111 (45.3%) out of 245 samples. Of these, 85 (34.7%) were smear positive on ZN staining and 102 (41.6%) were positive on LJ cultures. Rifampicin resistance was detected in 16 (6.5%) patients. Nine out of 19 pus samples (47.3%) were positive for MTB by Gene Xpert, 03 (15.8%) on ZN staining and 04 (21%) on LJ culture. MTB could not be detected in any other extrapulmonary sample. CONCLUSION: Xpert MTB/RIF is a sensitive method for rapid diagnosis of Tuberculosis, especially in smear negative cases and in EPTB as compared to the conventional ZN staining. Among EPTB cases the highest yield of positivity was shown in Pus samples. For countries endemic for TB GeneXpert can serve as a sensitive and time saving diagnostic modality for pulmonary and EPTB.

Concomitant presence of culture-proven active pulmonary tuberculosis in patients with chronic obstructive pulmonary disease - A hospital based study.

OBJECTIVE: To find out the prevalence of concomitant active pulmonary Tuberculosis (TB) in patients of Chronic Obstructive Pulmonary Disease (COPD) using the gold standard liquid and solid culture media for the detection of acid fast bacillus. METHODS: Eighty clinically and radiologically diagnosed cases of COPD of any severity, ≥40 years of age with no previous history of anti-tuberculous therapy were selected from department of Pulmonology, Jinnah Hospital, Lahore. Detailed demographic profile, clinical symptomatology and history of smoking were recorded. Sputum samples of these patients were subjected to ZiehlNeelsen (ZN) stain and culture on Lowenstein-Jensen (L.J) medium and Mycobacterium Growth Indicator Tube (MGIT) for the detection and isolation of Mycobacteriumtuberculosis (MTB). RESULTS: Out of 80 COPD patients, 6 (7.5%) were culture positive for acid fast bacillus consistent with active tuberculous infection. The concomitance was more prevalent in elderly, male, smokers. MGIT was a more sensitive and a rapid technique to detect the presence of mycobacterium as compared to LJ culture media and ZN stain. CONCLUSION: The prevalence of active TB in COPD patients was 7.5%. Detection was improved when liquid culture media was employed for the detection of acid fast bacillus. Regular monitoring and screening of patients with COPD for PTB should be routinely carried out in susceptible cohort to avoid cross spreading of infection and appropriate management.

GeneXpert Technology for the diagnosis of HIV-associated tuberculosis: Is scale-up worth it?

Recent evaluations of the GeneXpert MTB/RIF assay for the simultaneous detection of Mycobacterium tuberculosis and drug resistance in less than 2 h have stimulated tremendous enthusiasm. This is the breakthrough that tuberculosis (TB) control has been waiting for. In this (retrospective review) case study, sputum samples from strongly suspected pulmonary tuberculosis patients were collected and assessed for the GeneXpert MTB/RIF assay for diagnosing TB and drug resistance in comparison with other tests, including Ziehl-Neelsen smear and Löwenstein-Jensen test. Of 3,784 cases, 5.7% (216/3,784) were human immunodeficiency virus (HIV)-positive and TB co-infected patients. In diagnosing HIV-positive and TB co-infected cases, the sensitivity and specificity of GeneXpert were 76.4% and 100%. While in HIV-negative and TB suspected cases, the sensitivity and specificity were 95.6% and 100%. This new test represents a major milestone for global TB diagnosis and care. It also represents new hope for the millions of people who are at the highest risk of TB and drug-resistant disease. GeneXpert is World Health Organization-endorsed technology representing the gold standard for TB testing despite attaining less sensitivity for HIV and TB co-infected patients as compared to HIV-negative patients.

Distribution of blaCTX - M , blaTEM , blaSHV and blaOXA genes in Extended-spectrum-ß-lactamase-producing Clinical isolates: A three-year multi-center study from Lahore, Pakistan.

Background: Frequency of extended-spectrum-ß-lactamase-producing clinical isolates is increasing worldwide. This is a multi-center study which was aimed to check the frequency of third-generation cephalosporin resistance and distribution of the key genetic determinants of Extended-spectrum-ß-lactamase-producing Clinical isolates in Pakistan. Methods: A total of 2372 samples were processed in three tertiary care hospitals and one diagnostic research center of Lahore, Pakistan during Aug-2014 to Sep-2017. Analytical profile index (API 20-E) was used for biochemical characterization of isolates. Antibiotic susceptibility testing (AST) and third generation cephalosporin resistant (3GC-R) isolates were subjected to: double disc synergism test (DDST), combination disc test (CDST) and epsilometric test (E-test) for confirmation of ESBL-production. PCR amplification of isolates with plasmid and genomic DNA was performed. Amplicon sequences were checked for gene-variants and statistical analyses were performed to check the significance of data. Results: A total of 497/995 (50%) isolates including Escherichia coli 65% (n = 321), Klebsiella spp. 25% (n = 124) and Pseudomonas. 5% (n = 24), Enterobacter spp. 4% (n = 20) and Acinetobacter spp. 2% (n = 8) were screened as third generation cephalosporin resistant (3GC-R). Urine 56% (n = 278) followed by pus 20% (n = 99) and wound swab 6% (n = 29) were frequent sources. Incidence of ESBL-producers detected by combination disc test was 79% (n = 392). PCR revealed blaCTX - M (76%) gene followed by blaOXA (52%), blaTEM (28%) and blaSHV (21%) were most prevalent among ESBL-producers detected by CDST. blaCTX - M - 1(65%), blaOXA (78%) and blaTEM (57%) genes were carried on plasmids. Amplicon sequencing revealed blaCTX - M - 15 (75%), blaOXA - 1 (49%) and blaTEM - 1B (34%) and 21 (n = 28) isolates carried three genes in them. Conclusion: Prevalence of ESBL-producing isolates has increased 1.13 folds during study years. Isolates had high prevalence of ESBL-encoding blaCTXM - 15 gene and narrow spectrum blaOXA - 1 and blaTEM - 1B were also prevalent.

Pseudomonas aeruginosa: Evaluation of Pathogen Burden and Drug-Resistance Trends in a Tertiary Care Hospital.

OBJECTIVE: To evaluate the pathogen burden and antibiotic-resistance trends of Pseudomonas aeruginosa among hospitalised patients at a tertiary care hospital. STUDY DESIGN: Retrospective, hospital record-based, cross-sectional study. PLACE AND DURATION OF STUDY: Microbiology Laboratory, Allama Iqbal Medical College/Jinnah Hospital, Lahore, from January 2014 to December 2016. METHODOLOGY: A total of 5,960 samples were collected from clinically suspected cases of bacterial infections, admitted to the hospital. Microbial identification and antibiotic susceptibility pattern were carried out and analysed. RESULTS: Out of a total of 5,960 samples, Pseudomonas aeruginosa was isolated from 1,268 (21.2%) specimens. Department-wise isolation rate was n=600 (42.9%), n=268 (15.4%), n=201 (12.6%), and n=199 (16.0%) from intensive care unit (ICU), surgical units, medical units, and Gynae wards, respectively (p<0.0001). Sample-wise isolation rate was, wound swabs n=448 (35%), urine n=356 (28%), sputum n=187 (14 %), tracheal aspirate n=127 (10%), blood n=99 (7%), and broncho-alveolar lavage n=51 (4%) (p<0.0001). Drug-resistance pattern showed low rates for carbapenems(meropenem n=440 (35%), Imipenem n=436 (34%) and beta-lactam + beta-lactamase inhibitor combination (piperacillin+tazobactam n=437 (34%) while alarming rates were observed for cephalosporins (ceftazidime n=716 (56%), fluoroquinolones (ciprofloxacin n=690 (54%), cefoperazone+sulbactam n=685 (54%), aminoglycosides (gentamicin, n=669 (53%), amikacin n=608 (48%), and monobactams (aztreonam n=666 (52%). Decreasing trend was observed only for amikacin 63% to 37%, aztreonam showed similar pattern throughout, while there was an increasing trend of drug resistance in all groups of antibiotics. CONCLUSION: Emerging drug-resistant strains of Pseudomonas aeruginosa are probably linked to the injudicious use of antibiotics, leading to ineffective empirical therapy. Therefore, we suggest that culture and antimicrobial susceptibility testing should be done for targeted antimicrobial therapy against Pseudomonas aeruginosa.

High frequency and molecular epidemiology of metallo-ß-lactamase-producing gram-negative bacilli in a tertiary care hospital in Lahore, Pakistan.

Background: Metallo-ß-lactamase (MBL)-producing isolates have a strong impact on diagnostic and therapeutic decisions. A high frequency of MBL-producing gram-negative bacilli has been reported worldwide. The current study was based on determining the incidence of MBL-producing imipenem-resistant clinical isolates and investigating the ß-lactamase gene variants in strains conferring resistance to a carbapenem drug (imipenem). Methods: A total of 924 gram negative isolates were recovered from a tertiary care hospital in Lahore, Pakistan, during a two-year period (July 2015 to February 2017). The initial selection of bacterial isolates was based on antibiotic susceptibility testing. Strains resistant to imipenem were processed for the molecular screening of ß-lactamase genes. Statistical analysis for risk factor determination was based on age, gender, clinical specimen and type of infection. Results: The rate of imipenem resistance was calculated to be 56.51%. Among the 142 strains processed, the phenotypic tests revealed that the incidence of MBLs was 63.38% and 86.61% based on the combination disc test and the modified Hodge test, respectively. The frequencies of bla TEM, bla SHV, bla OXA, bla IMP-1, and bla VIM genes were calculated to be 46%, 34%, 24%, 12.5% and 7%, respectively. The co-expression of bla MBL (bla IMP and bla VIM) and bla ESBL (bla TEM, bla SHV, bla OXA) was also detected through multiplex and singleplex PCR. bla OXA, bla TEM and bla SHV coexisted in 82% of the isolates. Co-expression of ESBL and MBL genes was found in 7% of the isolates. Conclusion: To our knowledge, this is the first report from Pakistan presenting the concomitant expression of bla OXA, bla TEM and bla SHV with bla IMP-1 and bla VIM in MBL-producing gram-negative bacilli.

Prevalence of extended-spectrum-ß-lactamase-producing Enterobacteriaceae: first systematic meta-analysis report from Pakistan.

Background: South-Asia is known as a hub for multidrug-resistant (MDR) bacteria. Unfortunately, proper surveillance and documentation of MDR pathogens is lacking in Pakistan. The alarming increase in the prevalence of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae is a serious problem. From this perspective, we analysed published data regarding ESBL-producing Enterobacteriaceae in different regions of Pakistan. Methods: A meta-analysis was performed to determine the prevalence of ESBL-producing Enterobacteriaceae in Pakistan. A Web-based search was conducted in electronic databases, including PubMed, Scopus and PakMedi Net (for non-indexed Pakistani journals). Articles published (in either indexed or non-indexed journals) between January 2002 and July 2016 were included in the study. Relevant data were extracted, and statistical analysis was performed using the Metaprop command of STATA version 14.1. Results: A total of 68 studies were identified from the electronic data base search, and 55 of these studies met our inclusion criteria. Pakistan's overall pooled proportion of ESBL-producers was 0.40 (95% CI: 0.34-0.47). The overall heterogeneity was significant (I2 = 99.75%, p < 0.001), and significant ES = 0 (Z = 18.41, p < 0.001) was found. OXA, SHV, TEM and CTX-M were the most commonly found gene variants for ESBLs in these studies. Conclusion: The prevalence of ESBL-producing Enterobacteriaceae is high in Pakistan. Little is known about the annual frequency of ESBLs and their prevalence in different provinces of Pakistan. No data are available regarding ESBL frequency in Baluchistan. This underscores an urgent demand for regular surveillance to address this antimicrobial resistance problem. Surveillance to better understand the annual ESBL burden is crucial to improve national and regional guidelines.

False Negativity of Ziehl-Neelsen Smear Microscopy: Is the Scale-up the Worth It in Developing Countries?

OBJECTIVE: To evaluate the false negative results of Ziehl-Neelsen (ZN) smear microscopy. STUDY DESIGN: Descriptive study. PLACE AND DURATION OF STUDY: Mycobacteriology Laboratory, Allama Iqbal Medical College (AIMC) and Jinnah Hospital, Lahore (JHL), Pakistan, from February 2014 to August 2016. METHODOLOGY: A total of 3,951 (pulmonary 2,773 and extra-pulmonary 1,178) samples were collected from strong TB suspected patients attending JHL Lahore. Follow-up cases were excluded. Every specimen was processed for ZN smear microscopy, Lowenstein Jensen (LJ) culture. SPSS 21.0 was used; false negative and positive results of ZN smear were calculated keeping LJ culture as gold standard. RESULTS: Out of total 3,951 samples, sputum was most frequently found pulmonary sample 48.4% (n=1915), extra- pulmonary samples, pleural fluid and pus samples were most commonly observed samples 12.0% (n=476) and 8.3% (n=329), respectively. Overall false negativity was 23.1% (pulmonary=19.6%, extra-pulmonary=29.2%) (p<0.001), Maximum false negative results were observed in pericardial, synovial, pleural fluids, and pus samples as 40.0%, 38.0%, 33.0% and 32.0%, respectively. CONCLUSION: ZN smear microscopy is not a very efficient tool in case of patients with the low mycobacterial load. Therefore, National TB Control programs should consider extending their diagnostic approaches from ZN microscopy to more advanced techniques.

Gene variability and degree of expression of vaccine candidate factor H binding protein in clinical isolates of Neisseria meningitidis.

The factor H binding protein (fHbp) is currently being evaluated in clinical trials as a vaccine candidate for a meningococcal group B vaccine. We have previously described the prevalence and sequence variation of fHbp (Jacobsson et al., 2009) and here we investigate the expression of the antigen. The present study includes isolates from carriers (n = 62) and patients with invasive Neisseria meningitidis infections (n = 146), of which 62 had a fatal outcome. Among the invasive isolates from patients with fatal and non-fatal infections fHbp allele 1 was most common (42% and 29% respectively), but it was only identified in 3% of the carrier isolates, where allele 16 was most frequent (13%). The Fluorescence-activated cell sorting analysis identified fHbp expression in all except seven isolates and further analysis by Western blot showed that five of these seven samples were indeed negative using a polyclonal anti-fHbp serum. The negative isolates belonged to serogroup B fHbp allele 24, Y allele 104, and W-135 allele 16 (all invasive). Two were non-serogroupable carrier isolates (allele 21 and 101). An interesting finding is that isolates from invasive infections with fatal outcome had lower expression of fHbp or lower affinity for the fHbp antibody compared to isolates from non-fatal invasive infections and carriers.