Comparison of Two Specimen-Collection and Microscopy Methods for the Rapid Laboratory Diagnosis of Seborrheic Dermatitis- A Pilot Study

Malassezia spp. causes seborrheic dermatitis. For laboratory diagnosis, skin scrapings are collected and mounted in potassium hydroxide (KOH) for microscopy and processed for culture. Obtaining scrapings has disadvantages and KOH lacks colour contrast making interpretation difficult. This pilot study compared the results of specimen collection by cellophane tape method with scraping method. It also compared microscopy using Chicago Sky Blue 6B (CSB) stain plus KOH with the conventional method using KOH alone.METHODSSkin specimens were collected from the affected sites of 80 patients by scraping and cellophane tape. Specimens were subjected to KOH examination, KOH plus CSB stain, and culture for the presence of Malassezia spp.RESULTSA total of 160 specimens were collected from 80 patients for microscopy. Of 160 specimens, each was subjected to KOH and CSB plus KOH, 145 (91%) demonstrated Malassezia spp. by CSB plus KOH and 124 (77.5%) by KOH alone (p= 0.001). Cellophane tape method yielded 141 (88%) positive results compared to 128 (80%) by skin scraping (p=0.047). The odds of detecting Malassezia spp. was 4.4 times greater when the specimen was collected by cellophane tape and subjected to microscopy with CSB and KOH than when it was collected by scraping and examined microscopically with KOH alone (p= 0.002).CONCLUSIONSCellophane tape is a convenient method for specimen collection. CSB stain provides colour contrast and enables easy identification of fungal elements.

Prevalence of Intestinal Parasites in HIV Seropositive Patients Attending an Integrated Counselling and Testing Centre

HIV infection is the most disastrous and invariably fatal disease. Its devastating effect is due to its relentless and eventually complete destruction of the immune system. As a result, people infected with HIV die not due to the virus itself, but rather due to plethora of opportunistic infections that characterize AIDS. Intestinal parasitic infections are a significant cause of morbidity and mortality in patients infected with HIV in which Diarrhoea is one of the most common clinical presentations.1 With this background, a prospective study was carried out to determine the prevalence of intestinal parasites in HIV seropositive patients attending an Integrated Counselling Testing Centre (ICTC) of a tertiary care hospital.METHODSA cross-sectional study was conducted in a tertiary care multispecialty teaching hospital for a period of one year. Stool specimens of 250 HIV seropositive patients above 18 years of age and belonging to all genders, were screened for intestinal parasites in the present study. The stool specimens submitted were processed using direct wet mounts, concentration technique of formol ether, sedimentation and saturated common salt solution, and permanent staining techniques using Modified Acid-Fast staining, Trichrome staining and Modified Trichrome staining methods.RESULTSThe prevalence of intestinal parasites in HIV seropositive patients was found to be 27.6% (69/250). Protozoan parasites were predominant and were detected in 81.15% (56/69), followed by intestinal helminths in 11.59% (8/69) and coccidian parasites in 7.24% (5/69).CONCLUSIONSIntestinal parasites are a common source of infection in HIV seropositive patients. These patients are a threat not only to themselves but also to others in the community as well. Hence routine screening of all HIV seropositive patients is a must in order to prevent and reduce morbidity and mortality in the community.

Prevalence of pulmonary tuberculosis in patients with diabetes mellitus and lower respiratory tract infection

Background: Pulmonary Tuberculosis (PTB) still remains a global public health problem. Diabetes Mellitus (DM), is a metabolic disorder characterized by hyperglycaemia. Diabetes along with poor glycaemic control leads to an immune compromised state. As prevalence of both TB and DM is increasing in India, this association of PTB and DM may prove a threat to TB control program. Aims and objectives of the study was to detect prevalence of pulmonary tuberculosis in patients with DM and Lower Respiratory Tract Infection (LRTI).Methods: Sputum specimen from consecutive 250 known diabetic adult patients with type 2 diabetes and clinical evidence of LRTI were processed for microscopy, solid culture and Xpert MTB/RIF assay. Clinical findings, duration of DM, regularity of treatment and recent fasting blood glucose level were noted.Results: TB was detected in 31(12.8%) patients. Microscopy, culture and Xpert assay were positive in 14(5.6%), 29(11.6%) and 24(9.5%) cases respectively. Culture detected seven cases more than Xpert assay. Two additional cases were detected by Xpert assay than culture. Rifampicin resistance was detected in seven (29.17%) cases by Xpert assay. TB detection rate was higher in patients with more than two weeks of cough (14.38%), history of tuberculosis (15.9%), hyperglycemia (13.9%) and significantly higher in those with irregular anti-diabetic treatment (35.7%).Conclusions: Irregular anti-diabetic treatment, hyperglycaemia and history of tuberculosis were strongly associated with pulmonary TB. Xpert assay should be used as the initial diagnostic test for detection of tuberculosis as well as rifampicin resistance in diabetic patients by TB control programme.

Distribution of killer immunoglobulin-like receptor genes in HIV infected long-term non-progressors from Mumbai, India

Background: Few reports suggest the association of killer immunoglobulin-like receptors of natural killer cells with human immunodeficiency virus infection. India with world's third largest population of human immunodeficiency virus / acquired immunodeficiency syndrome, offers scope to study such association. Objective: Current study (2010-2015) was designed to evaluate if killer immunoglobulin-like receptors gene polymorphisms are associated with HIV infection outcomes specifically, with long term non progressors. Methods: Killer immunoglobulin-like receptors genotyping was done using polymerase chain reaction - sequence-specific primer method. Viral load was measured by Cobas Taqman HIV-1 test. Estimation of CD4 counts was done using BD FACS CD4 count reagent. Results: The activating gene frequencies identified were 3DS1 (53.8%), 2DS3 (69.2%), 2DS4 (76.9%), 2DS5 (69.2%), 2DS1 (76.9%) and 2DS2 (92.3%). The inhibitory gene frequencies were 2DL2 (92.3%), 2DL5 (76.9%), 2DL3 (69.5%), 3DL1 (84.6%), 3DL2 (92.3%) and 2DL1 (100%). The results highlight high frequency of 3DS1/3DL1 heterozygote and killer immunoglobulin-like receptor 2DS1, among these long term non progressors indicating their possible association with slow progression. Genotype analysis shows total 13 genotypes, of which 8 genotypes were identified for the first time from India. Two genotypes were unique/novel, which were unreported. All genotypes observed in this study were considered to be Bx genotype (100 %). Limitations: A small sample size (n=13, due to a rare cohort) and the absence of control group were the limitations of this study. Conclusions: The present study highlights the distribution of killer immunoglobulin-like receptor genes in a very rare group of human immunodeficiency virus -1 infected individuals - long term non progressors. All the long term non progressors tested show the presence of Bx haplotype and each long term non progressors has a different killer immunoglobulin-like receptor genotype.

Tricalcium phosphate solubilization and nitrogen fixation by newly isolated Aneurinibacillus aneurinilyticus CKMV1 from rhizosphere of Valeriana jatamansi and its growth promotional effect

Abstract Aneurinibacillus aneurinilyticus strain CKMV1 was isolated from rhizosphere of Valeriana jatamansi and possessed multiple plant growth promoting traits like production of phosphate solubilization (260 mg/L), nitrogen fixation (202.91 nmol ethylene mL-1 h-1), indole-3-acetic acid (IAA) (8.1 µg/mL), siderophores (61.60%), HCN (hydrogen cyanide) production and antifungal activity. We investigated the ability of isolate CKMV1 to solubilize insoluble P via mechanism of organic acid production. High-performance liquid chromatography (HPLC) study showed that isolate CKMV1 produced mainly gluconic (1.34%) and oxalic acids. However, genetic evidences for nitrogen fixation and phosphate solubilization by organic acid production have been reported first time for A. aneurinilyticus strain CKMV1. A unique combination of glucose dehydrogenase (gdh) gene and pyrroloquinoline quinone synthase (pqq) gene, a cofactor of gdh involved in phosphate solubilization has been elucidated. Nitrogenase (nif H) gene for nitrogen fixation was reported from A. aneurinilyticus. It was notable that isolate CKMV1 exhibited highest antifungal against Sclerotium rolfsii (93.58%) followed by Fusarium oxysporum (64.3%), Dematophora necatrix (52.71%), Rhizoctonia solani (91.58%), Alternaria sp. (71.08%) and Phytophthora sp. (71.37%). Remarkable increase was observed in seed germination (27.07%), shoot length (42.33%), root length (52.6%), shoot dry weight (62.01%) and root dry weight (45.7%) along with NPK (0.74, 0.36, 1.82%) content of tomato under net house condition. Isolate CKMV1 possessed traits related to plant growth promotion, therefore, could be a potential candidate for the development of biofertiliser or biocontrol agent and this is the first study to include the Aneurinibacillus as PGPR.

Molecular Cloning and Sequencing of AlkalophilicCellulosimicrobium cellulans CKMX1 Xylanase Gene Isolated from Mushroom Compost and Characterization of the Gene Product

ABSTRACT A xylanolytic bacterium was isolated from mushroom compost by using enrichment technique. Results from the metabolic fingerprinting, whole-cell fatty acids methyl ester analysis and 16S rDNA sequencing suggested the bacterium to be Cellulosimicrobium cellulans CKMX1. Due to the xylanolytic activity of this bacterium, isolation and characterization of the xylanase gene were attempted. A distinct fragment of about 1671 bp was successfully amplified using PCR and cloned into Escherichia coli DH5α. A BLAST search confirmed that the DNA sequence from the amplified fragment was endo-1, 4-beta-xylanase, which was a member of glycoside hydrolase family 11. It showed 98% homology withCellulosimicrobium sp. xylanase gene (Accession no. FJ859907.1) reported from the gut of Eisenia fetida in Korea. In silicophysico-chemical characterization of amino acid sequence of xylanase showed an open reading frame encoding a 556 amino acid sequence with a molecular weight of 58 kDa and theoretical isolectric point (pI) of 4.46 was computed using Expasy's ProtParam server. Secondary and homology based 3D structure of xylanase was analysed using SOPMA and Swiss-Prot software.

Utility of MPT 64 antigen detection assay for rapid characterization of mycobateria in a resource constrained setting.

Introduction: Important reasons for the negligible numbers of laboratories performing characterization of Mycobacteria in resource constrained settings are requirement of biosafety measures, longer turnaround time and laborious nature of tests. A rapid, accurate and simple test for characterization is required. “SD BIOLINE TB Ag MPT 64 Rapid®” is a rapid immunochromatographic test for differentiation of Mycobacteria into M. tuberculosis Complex (MTBC) and nontuberculous mycobacteria (NTM). Aim: To evaluate a commercial assay, SD TB Ag MPT64 Rapid® for characterization of Mycobacteria isolated on Lowenstein Jensen (LJ) medium. Material and methods: 150 non duplicate isolates which were previously characterized as MTBC or NTM based on standard phenotypic characteristics were tested by the commercial assay after blinding. The result of the conventional phenotypic test and the commercial assay was compared. Any discordant result was referred for confirmation by genotypic Mycobacterium CM assay (Hain’s life sciences, Germany). Sensitivity and specificity of the commercial assay was calculated using the results of conventional phenotypic and genotypic tests as gold standard. Results: Phenotypically, 124 isolates were characterized as MTBC and 26 as NTM. The commercial assay gave concordant results for 149 isolates. One MTBC isolate did not demonstrate a band. The sensitivity, specificity, PPV and NPV was 99.19%, 100 %, 100% and 97.3% respectively. The total turnaround time for the rapid assay was 30 minutes compared to a few hours to days for phenotypic and genotypic method. Conclusion: “SD BIOLINE TB Ag MPT 64 Rapid®” is a simple, rapid and reliable test to differentiate MTBC from NTM.

Incremental yield in sputum smear positivity by examining a second early morning sputum specimen in follow-up patients on dots: 7 year analysis of RNTCP laboratory register.

Background: Patients receiving DOTS undergo periodic follow-up sputum examination, which aids in monitoring response to treatment. Continued or new smear positivity at follow up examination entails extension of intensive phase or change in treatment category and the need for culture and drug susceptibility test. Setting: Tuberculosis microscopy centre at a tertiary care teaching hospital, Mumbai, India. Objective: To determine the incremental yield in sputum smear positivity by examining a second early morning sputum specimen in follow-up patients on DOTS. Design: Retrospective analysis of follow up sputum microscopy results recorded in tuberculosis laboratory register for the period 2002-2008. Results: During the study period, 5015 follow-up patients submitted two early morning sputum specimens, of which 501(9.99 %) patients were detected to be smear-positive. Out of smear positive patients under study, 324 patients had both specimens positive, 79 patients had only first specimen positive and 98 patients had only second specimen positive. The incremental yield was 1.95 % of total and 19.5 % of smear positives. Conclusion: Discordant smears were present in nearly a third of patients detected smear positive during follow-up. More than half of these patients were detected only by examining second specimen. The incremental yield by examining the second early morning specimen was 1.95 % of total and 19.5 % of smear positive specimens. It is important to detect each possible smear positive follow-up patient as they are likely to benefit from altered treatment. The inclusion of a second early morning sputum specimen examination is essential to maximize their detection.

Utility of Microscopic Observation of Drug Susceptibility (MODS) assay for mycobacterium tuberculosis in resource constrained settings.

Introduction: Multidrug Resistant Tuberculosis (MDR TB) is a global health problem. Conventional techniques or automated systems for diagnosis and drug susceptibility testing are either comparatively slow or costly. Microscopic Observation Drug Susceptibility [MODS] assay is a simultaneous detection and direct drug susceptibility test [DST] method which relies on the characteristic growth of Mycobacterium tuberculosis (MTB) in a liquid medium. Aim: Comparison between MODS assay and culture on Lowenstein Jensen (LJ) medium with respect to; i) detection of mycobacterial growth and time taken for culture positivity (ii) to compare concordance of susceptibility results of MTB isolate by MODS with proportion method using LJ medium. Method: A prospective study was carried out on 171 acid fast smear positive sputum specimens. The decontaminated sediment was used for culture and DST using LJ medium (proportion method) and MODS assay plates containing supplemented Middlebrook 7H9 broth with and without critical concentrations of isoniazid and rifampicin. Results: Median time to growth and DST using MODS assay was 10 days and that with LJ medium was 24 and 66 days respectively. The sensitivity and specificity of MODS assay was 100%. All the isolates were characterized as MTB. MODS demonstrated 98.8% and 99.4% concordance for isoniazid and rifampicin respectively and 100% for MDRTB. Positive and negative predictive value for MDRTB was 100%. Conclusion: MODS assay offers a rapid, simple, economical and feasible method for simultaneous culture and DST of MTB. Utility of MODS needs to be ascertained in extrapulmonary TB cases.