Synthesis, evaluation, molecular docking, and molecular dynamics studies of novel N-(4-[pyridin-2-yloxy]benzyl)arylamine derivatives as potential antitubercular agents.

A new series of novel triclosan (2,4,4'-trichloro-2'-hydroxydiphenylether) analogues were designed, synthesized, and screened for their in vitro antimycobacterial and antibacterial activities. Most of the compounds showed significant activity against Mycobacterium tuberculosis H37Rv strain with minimum inhibitory concentration (MIC) values in 20-40 µM range in GAST/Fe medium when compared with triclosan (43 µM) in the first week of assay, and after additional incubation, seven compounds, that is, 2a, 2c, 2g, 2h, 2i, 2j, and 2m, exhibited MIC values at the concentration of 20-40 µM. The compounds also showed more significant activity against Bacillus subtilis and Staphylococcus aureus. The synthesized compounds showed druggable properties, and the predicted ADME (absorption, distribution, metabolism, and excretion) properties were within the acceptable limits. The in silico studies predicted better interactions of compounds with target protein residues and a higher dock score in comparison with triclosan. Molecular dynamics simulation study of the most active compound 2i was performed in order to further explore the stability of the protein-ligand complex and the protein-ligand interaction in detail.

In silico studies, synthesis and anticancer activity of novel diphenyl ether-based pyridine derivatives.

A series of novel 2-amino-4-(3-hydroxy-4-phenoxyphenyl)-6-(4-substituted phenyl) nicotinonitriles were synthesized and evaluated against HepG2, A-549 and Vero cell lines. Compounds 3b (IC50 16.74 ± 0.45 µM) and 3p (IC50 10.57 ± 0.54 µM) were found to be the most active compounds against A-549 cell line among the evaluated compounds. Further 3b- and 3p-induced apoptosis was characterized by AO/EB (acridine orange/ethidium bromide) nuclear staining method and also by DNA fragmentation study. A decrease in cell viability and initiation of apoptosis was clearly evident through the morphological changes in the A-549 cells treated with 3b and 3p when stained with this method. Fragmentation of DNA into nucleosomes was observed which further confirmed the cell apoptosis in cells treated with compound 3b. Flow cytometry studies confirmed the cell cycle arrest at G2/M phase in A549 cells treated with compound 3b. Further in silico studies performed supported the in vitro anticancer activity of these compounds as depicted by dock score and binding energy values.

Assessment for learning: a needs analysis study using formative assessment to evaluate the need for curriculum reform in basic sciences.

A needs analysis study for curriculum reform in basic sciences was conducted at Melaka Manipal Medical College, India, by means of a formative assessment method, namely Basic Science Retention Examination (BSRE). Students participated in a BSRE, which comprised recall and clinical multiple-choice questions in six discipline areas. They also rated the clinical relevance of each question and provided responses to three open-text questions about the exam. Pass rates were determined; clinical relevance ratings and performance scores were compared between recall type and clinical questions to test students' level of clinical application of basic science knowledge. Text comments were thematically analyzed to identify recurring themes. Only one-third of students passed the BSRE (32.2%). Students performed better in recall questions compared with clinical questions in anatomy (51.0 vs. 40.2%), pathology (45.1 vs. 38.1%), pharmacology (41.8 vs. 31.7%), and biochemistry (43.5 vs. 26.9%). In physiology, students performed better in clinical questions compared with the recall type (56.2 vs. 45.8%). Students' response to BSRE was positive. The findings imply that transfer of basic science knowledge was poor, and that assessment methods should emphasize clinical application of basic science knowledge.

Diversity, in-vitro virulence traits and antifungal susceptibility pattern of gastrointestinal yeast flora of healthy poultry, Gallus gallus domesticus.

BACKGROUND: Poultry farming and consumption of poultry (Gallus gallus domesticus) meat and eggs are common gastronomical practices worldwide. Till now, a detailed understanding about the gut colonisation of Gallus gallus domesticus by yeasts and their virulence properties and drug resistance patterns in available literature remain sparse. This study was undertaken to explore this prevalent issue. RESULTS: A total of 103 specimens of fresh droppings of broiler chickens (commercial G domesticus) and domesticated chickens (domesticated G domesticus) were collected from the breeding sites. The isolates comprised of 29 (33%) Debaryozyma hansenii (Candida famata), 12 (13.6%) Sporothrix catenata (C. ciferrii), 10 (11.4%) C. albicans, 8 (9.1%) Diutnia catenulata (C. catenulate), 6 (6.8%) C. tropicalis, 3 (3.4%) Candida acidothermophilum (C. krusei), 2 (2.3%) C. pintolopesii, 1 (1.1%) C. parapsilosis, 9 (10.2%) Trichosporon spp. (T. moniliiforme, T. asahii), 4 (4.5%) Geotrichum candidum, 3 (3.4%) Cryptococcus macerans and 1 (1%) Cystobasidium minuta (Rhodotorula minuta). Virulence factors, measured among different yeast species, showed wide variability. Biofilm cells exhibited higher Minimum Inhibitory Concentration (MIC) values (µg/ml) than planktonic cells against all antifungal compounds tested: (fluconazole, 8-512 vs 0.031-16; amphotericin B, 0.5-64 vs 0.031-16; voriconazole 0.062-16 vs 0.062-8; caspofungin, 0.062-4 vs 0.031-1). CONCLUSIONS: The present work extends the current understanding of in vitro virulence factors and antifungal susceptibility pattern of gastrointestinal yeast flora of G domesticus. More studies with advanced techniques are needed to quantify the risk of spread of these potential pathogens to environment and human.

Distribution and Relationships of Antimicrobial Resistance Determinants among Extended-Spectrum-Cephalosporin-Resistant or Carbapenem-Resistant Escherichia coli Isolates from Rivers and Sewage Treatment Plants in India.

To determine the distribution and relationship of antimicrobial resistance determinants among extended-spectrum-cephalosporin (ESC)-resistant or carbapenem-resistant Escherichia coli isolates from the aquatic environment in India, water samples were collected from rivers or sewage treatment plants in five Indian states. A total of 446 E. coli isolates were randomly obtained. Resistance to ESC and/or carbapenem was observed in 169 (37.9%) E. coli isolates, which were further analyzed. These isolates showed resistance to numerous antimicrobials; more than half of the isolates exhibited resistance to eight or more antimicrobials. The blaNDM gene was detected in 14/21 carbapenem-resistant E. coli isolates: blaNDM-1 in 2 isolates, blaNDM-5 in 7 isolates, and blaNDM-7 in 5 isolates. The blaCTX-M gene was detected in 112 isolates (66.3%): blaCTX-M-15 in 108 isolates and blaCTX-M-55 in 4 isolates. We extracted 49 plasmids from selected isolates, and their whole-genome sequences were determined. Fifty resistance genes were detected, and 11 different combinations of replicon types were observed among the 49 plasmids. The network analysis results suggested that the plasmids sharing replicon types tended to form a community, which is based on the predicted gene similarity among the plasmids. Four communities each containing from 4 to 17 plasmids were observed. Three of the four communities contained plasmids detected in different Indian states, suggesting that the interstate dissemination of ancestor plasmids has already occurred. Comparison of the DNA sequences of the blaNDM-positive plasmids detected in this study with known sequences of related plasmids suggested that various mutation events facilitated the evolution of the plasmids and that plasmids with similar genetic backgrounds have widely disseminated in India.

Diagnostic validation of selected serological tests for detecting scrub typhus.

Clinical diagnosis of scrub typhus is often difficult because the symptoms are very similar to those of other febrile illness such as dengue, leptospirosis, malaria and other viral hemorrhagic fevers. Though better diagnostic tests are available for rickettsial diseases and scrub typhus elsewhere, the Weil-Felix test is still commonly used in India, mainly because microimmunofluorescence assays (M-IFA) were not available in India till recently and relevant staff had insufficient training. The present study was performed to investigate the performance of M-IFA, IgM ELISA, and Weil-Felix test on 546 non-repeated serum samples from subjects suspected of having scrub typhus. One hundred and forty-three of these 546 samples were positive by M-IFA; these cases were also confirmed clinically to have scrub typhus based on their dramatic responses to doxycycline therapy. IgM ELISA was positive in 122 of the 143 M-IFA positive cases and the Weil-Felix test in 96. Though the Weil-Felix test is a heterophile agglutination test, it was found in this study to have good specificity but far too little sensitivity to use as a routine diagnostic test. IgM ELISA can be a good substitute for M-IFA. Incorporation of multiple prototype antigens on M-IFA slides is likely one of the reasons for its superior performance. As newer and better diagnostic assays become available for scrub typhus diagnosis in developed countries, it will be imperative to also use such tests in other endemic countries to prevent over- or under-diagnosis of scrub typhus.

Impact of wastewater from different sources on the prevalence of antimicrobial-resistant Escherichia coli in sewage treatment plants in South India.

The sewage treatment plant (STP) is one of the most important interfaces between the human population and the aquatic environment, leading to contamination of the latter by antimicrobial-resistant bacteria. To identify factors affecting the prevalence of antimicrobial-resistant bacteria, water samples were collected from three different STPs in South India. STP1 exclusively treats sewage generated by a domestic population. STP2 predominantly treats sewage generated by a domestic population with a mix of hospital effluent. STP3 treats effluents generated exclusively by a hospital. The water samples were collected between three intermediate treatment steps including equalization, aeration, and clarification, in addition to the outlet to assess the removal rates of bacteria as the effluent passed through the treatment plant. The samples were collected in three different seasons to study the effect of seasonal variation. Escherichia coli isolated from the water samples were tested for susceptibility to 12 antimicrobials. The results of logistic regression analysis suggest that the hospital wastewater inflow significantly increased the prevalence of antimicrobial-resistant E. coli, whereas the treatment processes and sampling seasons did not affect the prevalence of these isolates. A bias in the genotype distribution of E. coli was observed among the isolates obtained from STP3. In conclusion, hospital wastewaters should be carefully treated to prevent the contamination of Indian environment with antimicrobial-resistant bacteria.

Acute kidney injury in scrub typhus.

BACKGROUND: We studied the urinary abnormalities and acute kidney injury (AKI) as per RIFLE criteria in scrub typhus. METHODS: A prospective case record-based study of scrub typhus was carried out from January 2009 to December 2010 in a tertiary hospital in South India. Patients were followed up until renal recovery or for at least 3 months after discharge. Univariate, chi-squared tests and multivariate logistic regression analyses were performed to identify the predictors of AKI. RESULTS: Scrub typhus was diagnosed in 259 patients. Urinary abnormalities were seen in 147 patients (56.7%) with 60 patients (23.2%) having AKI. All AKI patients had urinary abnormalities and 17 (28.3%) were oliguric. Applying RIFLE (risk, injury, failure, loss, end-stage kidney disease) criteria, R, I, F were present in 23 (38.33%), 13 (21.67%), and 24 patients (40%), respectively. Creatine phosphokinase (CPK) was raised in 33 patients (55%) and hemodialysis was required in 6 patients (10%). The case fatality rate in this study was 2 out of 259 (0.77%), both having AKI and others recovering clinically. Significant predictors of AKI were tachycardia [odds ratio (OR) 2.28], breathlessness (OR 2.281), intensive care requirement (OR 2.43), mechanical ventilation (OR 3.33), thrombocytopenia (OR 2.90) and CPK>80 U/L (OR 1.76) by univariate analysis and intensive care requirement (adjusted OR 2.89) and thrombocytopenia (AOR 2.28) by multivariable logistic regression. CONCLUSION: Scrub typhus should be part of the differential diagnosis of acute febrile illness with AKI. AKI in scrub typhus is usually mild, non-oliguric, and renal recovery occurs in most patients. Rhabdomyolysis may be contributory to AKI. Thrombocytopenia and intensive care requirement are significant predictors of AKI in scrub typhus.

Clostridium difficile infection at a tertiary care hospital in south India.

OBJECTIVE: The objective of this study was to detect C. difficile in patients presenting with Antibiotic Associated Diarrhoea. METHODS: Stool samples from twenty-five patients collected over a period of four months were processed for C. difficile by culture and the isolates were identified following standard methods. C. difficile toxins A and B and C. perfringens enterotoxin were detected by ELISA performed directly on stool specimens. RESULTS: Four patients (16%) were found positive for C. difficile infection. All patients with C. difficile infection received prior treatment with third-generation cephalosporins or beta-lactam/beta-lactamase inhibitor antibiotics. C. perfringens enterotoxin was found in two (8%) patients. Severe colitis was seen in one (25%) of the four patients who had co-infection with C. difficile and C. perfringens. CONCLUSION: This study demonstrated a significant occurrence of C. difficile infection in this hospital population. There is a need to further evaluate the role of C. perfringens in causing antibiotic associated diarrhoea. Good clinical and laboratory studies to generate local epidemiological data are essential to increase awareness among the treating clinicians about C. difficile infection. Also limited and rational use of broad spectrum antibiotics is recommended.

Significance of interleukin-6 (IL-6) and C-reactive protein (CRP) in children and young adults with febrile neutropenia during chemotherapy for cancer: a prospective study.

OBJECTIVES: We evaluated the usefulness of interleukin-6 (IL-6) and C-reactive protein (CRP) at the onset of febrile neutropenia and 72 hours later, in identifying risk groups and assessing response to antibiotic therapy. METHODS: All episodes of febrile neutropenia were divided in 3 study groups-microbiologically documented infection (MDI), clinically documented infection (CDI), and fever of unknown origin (FUO). Three outcome groups were defined as those responding to first-line antibiotics (R1), those responding to second-line antibiotics (R2), and those requiring antifungal therapy (RAF). Median values of IL-6 and CRP were compared between the groups. RESULTS: There were 57 episodes of febrile neutropenia among 26 patients younger than 25 years during 1 year of study period. On day 1, median IL-6 level was significantly lower in FUO group compared with CDI+MDI groups combined (P < 0.001). Rise in CRP on day 3 was highly significant to differentiate MDI group from other 2 groups (P < 0.001). The CRP also increased significantly on day 3 in RAF (P < 0.001) and R2 (P = 0.002) groups than in R1 group. CONCLUSIONS: Low level of IL-6 may help differentiate patients with FUO from those with documented infections. A rising CRP is indicative of serious infection.

Can tissue PCR augment the diagnostic accuracy in genitourinary tract tuberculosis?

PURPOSE: Conventional methods like smear and culture for Mycobacterium tuberculosis are of limited sensitivity and specificity. Histopathological examination (HPE) for the tissues obtained gives inconclusive diagnosis in the absence of caseous necrosis or stained acid-fast bacilli. This study was conducted to determine the utility of tissue PCR for diagnosing tuberculosis of the genitourinary tract (GUTB) and its comparative evaluation with HPE. PATIENTS AND METHODS: A prospective study was conducted from January 2006 to August 2009 with 78 tissue specimens (renal, prostate, epididymis, penile and soft tissue) from patients with clinically suspected GUTB. All the samples were processed for both PCR and histopathology. RESULTS: In 68 (87.1%) samples, results for both PCR and HPE were coinciding. False positivity and false negativity was observed in 5.1% (4/78) and 7.6% (6/78) samples, respectively. With HPE as the gold standard, PCR has shown sensitivity of 87.5% (95% CI 80.1; 91.9) and specificity of 86.7% (95% CI 74.9; 93.8) and positive agreement between two tests was observed as significant (0.7). PCR results were obtained within a mean period of 3.4 days while those of HPE were obtained in 7.2 days. CONCLUSIONS: Tissue PCR is a sensitive and specific method for obtaining early and timely diagnosis of GUTB. Application of tissue PCR results can augment the diagnostic accuracy in histopathologically labelled granulomatous inflammations.

Role of risk factors and socio-economic status in pulmonary tuberculosis: a search for the root cause in patients in a tertiary care hospital, South India.

OBJECTIVE: To determine the frequency of underlying risk factors and the socio-economic impact based on occupation in the development of tuberculosis. METHOD: Retrospective analysis of 207 clinically and microbiologically diagnosed patients with pulmonary tuberculosis (PTB) admitted to Kasturba Hospital in 2005 and 2006. Demographic details and underlying risk factors were statistically evaluated. RESULTS: Diabetes mellitus (DM) (30.9%) was the most prevalent condition and significantly more common than other risk factors like smoking (16.9%), alcoholism (12.6%), HIV (10.6%), malignancy (5.8%), chronic liver diseases (3.9%), history of contact with TB (3.4%), chronic corticosteroid therapy (2.9%), chronic kidney diseases and malnourishment (1.5%). There were 82 patients (39.6%) with no underlying risk factor. Men (M:F = 3.7:1) and patients older than 40 years had a higher incidence of co-existing conditions. PTB was significantly more common in blue-collar (44%) and white-collar (27.1%) workers than household workers (12.1%), students (10.6%) and retired/unemployed people (6.3%). CONCLUSION: Pulmonary tuberculosis had a significant impact and predominated in male patients co-existing with DM. Patients with DM and suggestive pulmonary symptoms should be screened for tuberculosis. More stringent health education and awareness programme should be implemented at the grass root level.

Salivary IL-6 levels in oral leukoplakia with dysplasia and its clinical relevance to tobacco habits and periodontitis.

The development of oral cancer proceeds through discrete molecular changes that are acquired from loss of genomic integrity after continued exposure to environmental risk factors. It is preceded in the majority of cases by clinically evident oral potentially malignant disorders, the most common of which is leukoplakia. Early detection of these oral lesions by screening methods using suitable markers is critical as it mirrors molecular alterations, long before cancer phenotypes are manifested. Assessment of salivary interleukin-6 (IL-6) as a marker of malignant progression was undertaken in patients with leukoplakia having coexisting periodontitis (n = 20), periodontitis patients without leukoplakia (n = 20), and healthy controls (n = 20) by competitive enzyme-linked immunosorbent assay. Results showed elevation of IL-6 levels in leukoplakia with coexisting periodontitis and in periodontitis patients when compared to healthy control (P < 0.001). Within the leukoplakia group, IL-6 level was found to be increased with increase in the severity of dysplasia. The use of tobacco was seen to play a significant role in the elevation of salivary IL-6.The importance of IL-6 as a specific marker for leukoplakia with dysplasia and the role of tobacco as an independent risk factor has been highlighted.

Detection and characterization of ESBL-producing Enterobacteriaceae from the gut of subsistence farmers, their livestock, and the surrounding environment in rural Nepal.

The increasing trend of gut colonization by extended-spectrum ß-lactamase (ESBL) producing Enterobacterales has been observed in conventional farm animals and their owners. Still, such colonization among domesticated organically fed livestock has not been well studied. This study aimed to determine the gut colonization rate of ESBL-producing Enterobacteriaceae and carbapenemase-producing Enterobacteriaceae (CPE) among rural subsistence farming communities of the Kaski district in Nepal. Rectal swabs collected by systematic random sampling from 128 households of subsistence farming communities were screened for ESBL-producing Enterobacteriaceae and CPE by phenotypic and molecular methods. A total of 357 (57%) ESBL-producing Enterobacteriaceae isolates were obtained from 626 specimens, which included 97 ESBL-producing Enterobacteriaceae (75.8%) from 128 adult humans, 101 (79.5%) from 127 of their children, 51 (47.7%) from 107 cattle, 26 (51%) from 51 goats, 30 (34.9%) from 86 poultry and 52 (42%) from 127 environmental samples. No CPE was isolated from any of the samples. blaCTX-M-15 was the most predominant gene found in animal (86.8%) and human (80.5%) isolates. Out of 308 Escherichia coli isolates, 16 human and two poultry isolates were positive for ST131 and were of clade C. Among non-cephalosporin antibiotics, the resistance rates were observed slightly higher in tetracycline and ciprofloxacin among all study subjects. This is the first one-health study in Nepal, demonstrating the high rate of CTX-M-15 type ESBL-producing Enterobacteriaceae among gut flora of subsistence-based farming communities. Gut colonization by E. coli ST131 clade C among healthy farmers and poultry birds is a consequential public health concern.

Bacterial organisms and antimicrobial resistance patterns.

Gram negative bacilli are the commonest cause of infections at all sites in the Indian setting. There is a considerable degree of resistance to both first line and second line gram negative agents. Resistance to gram positive cocci is less of a problem than that of gram negative bacilli. MRSA prevalence though is not insignificant.

Detection and characterization of ESBL-producing Enterobacteriaceae from the gut of healthy chickens, Gallus gallus domesticus in rural Nepal: Dominance of CTX-M-15-non-ST131 Escherichia coli clones.

The surge in the prevalence of drug-resistant bacteria in poultry is a global concern as it may pose an extended threat to humans and animal health. The present study aimed to investigate the colonization proportion of extended-spectrum ß-lactamase (ESBL) and carbapenemase-producing Enterobacteriaceae (EPE and CPE, respectively) in the gut of healthy poultry, Gallus gallus domesticus in Kaski district of Western Nepal. Total, 113 pooled rectal swab specimens from 66 private household farms and 47 commercial poultry farms were collected by systematic random sampling from the Kaski district in western Nepal. Out of 113 pooled samples, 19 (28.8%) samples from 66 backyard farms, and 15 (31.9%) from 47 commercial broiler farms were positive for EPE. Of the 38 EPE strains isolated from 34 ESBL positive rectal swabs, 31(81.6%) were identified as Escherichia coli, five as Klebsiella pneumoniae (13.2%), and one each isolate of Enterobacter species and Citrobacter species (2.6%). Based on genotyping, 35/38 examined EPE strains (92.1%) were phylogroup-1 positive, and all these 35 strains (100%) had the CTX-M-15 gene and strains from phylogroup-2, and 9 were of CTX-M-2 and CTX-M-14, respectively. Among 38 ESBL positive isolates, 9 (23.7%) were Ambler class C (Amp C) co-producers, predominant were of DHA, followed by CIT genes. Two (6.5%) E. coli strains of ST131 belonged to clade C, rest 29/31 (93.5%) were non-ST131 E. coli. None of the isolates produced carbapenemase. Twenty isolates (52.6%) were in-vitro biofilm producers. Univariate analysis showed that the odd of ESBL carriage among commercial broilers were 1.160 times (95% CI 0.515, 2.613) higher than organically fed backyard flocks. This is the first study in Nepal, demonstrating the EPE colonization proportion, genotypes, and prevalence of high-risk clone E. coli ST131 among gut flora of healthy poultry. Our data indicated that CTX-M-15 was the most prevalent ESBL enzyme, mainly associated with E. coli belonging to non-ST131clones and the absence of carbapenemases.

Adequacy of two versus three sputum specimens for the diagnosis of pulmonary tuberculosis.

BACKGROUND: The new guidelines issued by the Revised National Tuberculosis Control Programme for diagnosis of smear-positive tuberculosis recommend examination of only 2 sputum smears. We did a retrospective analysis of data from a designated microscopy centre to ascertain the diagnostic yield of 2 smears and the additional yield provided by the third smear. METHODS: Data were obtained from the designated microscopy centre attached to our medical college. A total of 3257 patients with suspected tuberculosis had undergone sputum examination between September 2004 and March 2009. However, only 1762 of them had 3 sputum specimens examined. Data were entered and analysed using SPSS version 11.5. RESULTS: Among the 1762 suspected patients, positivity in any 2 samples was found to be 17.7% while 19% were found to be positive in a single smear. A statistically insignificant association was found between the grading and positivity of the sputum samples using McNemar test. A positive third sample was found in 309 patients. If the first 2 samples were negative, the possibility of missing a third positive sample was 0.4%. CONCLUSION: Under field conditions, 2 sputum smears are as effective as 3 smears for diagnosing smear-positive tuberculosis.

Pulmonary nocardiosis due to Nocardia farcinica in a renal transplant recipient.

Nocardia farcinica is an infrequent cause of nocardiosis among the renal transplant recipients and it has not been reported so far from India. We report a case of pulmonary nocardiosis due to N. farcinica in a 32-year-old woman with hypothyroidism and post-renal transplant status, currently on immunosuppressive therapy (prednisolone, azathioprine and tacrolimus). The N. farcinica isolate was susceptible to trimethoprim-sulfamethoxazole (TMP-SMZ), linezolid, imipenem, gentamicin but resistant to ceftriaxone, ciprofloxacin, tobramycin, erythromycin, amoxycillin-clavulanic acid and tetracycline. Treatment with TMP-SMZ and linezolid resulted in marked clinico-radiological improvement but after two weeks both of the drugs had to be stopped due to severe pancytopenia as adverse effect of their use. Currently, the patient is on imipenem and remains stable after four weeks of treatment. In N. farcinica infections, multi antibiotic resistance and toxicity of some specific drugs enhances the risk of therapeutic failure in renal transplant recipients.

Comparative evaluation of two cold staining methods with the Ziehl-Neelsen method for the diagnosis of tuberculosis.

In developing countries pulmonary tuberculosis is usually diagnosed by detecting acid-fast bacilli (AFB) in sputum using a Ziehl-Neelsen (Z-N) staining method. However, in the field the traditional method of staining is difficult to carry out. This study evaluates the efficiency of two cold staining methods, namely Gabbet's and a modified two reagent cold staining method compared with the Z-N taken as the gold standard. Triplicate smears were prepared from 267 sputum samples and stained by the Z-N, Gabbet's, and a modified cold staining method, the smears were positive for AFB in 21 (7.87%), 18 (6.74%) and 19 (7.12%), respectively. The sensitivities for the Gabbet's and modified cold stain were 85.7% and 90.5%, respectively. The positive agreement between the Z-N and Gabbet's (92.3%) and Z-N and modified cold stain (95%) were good. The modified cold staining method was less time consuming and easier to perform in the field.