A Comparative Study of Serum, Urine and Saliva Using rk39 Strip for the Diagnosis of Visceral Leishmaniasis

Background: Immunochromatographic based rk39 antibody detection test became popular for the diagnosis of vis­ceral leishmaiasis (VL) because of high sensitivity, rapidity, easy to interpret, and cost effectiveness. However, false positive result after complete cure of the patients is the major limitation with this test. The aim of the study to access the usefulness of non-invasive samples i.e. urine and saliva by rk39 test for the diagnosis of visceral leishmaniasis in comparison to serum. Methods: Seventy two clinically suspected VL patients were enrolled in the study among which 61 cases were con­firmed as VL and 11 cases were included in the control group. Serum, urine, and saliva samples of all the cases were tested for rk39 dip stick test. Results: Urine and saliva both were equally sensitive as serum for the diagnosis kala-azar. In the control group, rk39 antibody test was negative in 10 cases out 11 (91%) with saliva in comparison to 4 cases with serum (36%), thereby found to be more specific. Conclusion: Saliva sample found to be highly reliable for the diagnosis of VL cases by rk39 test. The test with saliva sample showed less false positive result in comparison to serum sample, thereby can be used an adjunct with serum sample for the diagnosis of kala-azar in endemic areas

Two-dimensional difference gel electrophoresis (DIGE) analysis of sera from visceral leishmaniasis patients

Abstract Introduction Visceral leishmaniasis is a parasitic infection caused by Lesihmania donovani complex and transmitted by the bite of the phlebotomine sand fly. It is an endemic disease in many developing countries with more than 90% of the cases occurring in Bangladesh, India, Nepal, Sudan, Ethiopia and Brazil. The disease is fatal if untreated. The disease is conventionally diagnosed by demonstrating the intracellular parasite in bone marrow or splenic aspirates. This study was carried out to discover differentially expressed proteins which could be potential biomarkers. Methods Sera from six visceral leishmaniasis patients and six healthy controls were depleted of high abundant proteins by immunodepletion. The depleted sera were compared by 2-D Difference in gel electrophoresis (DIGE). Differentially expressed proteins were identified the by tandem mass spectrometry. Three of the identified proteins were further validated by western blotting. Results This is the first report of serum proteomics study using quantitative Difference in gel electrophoresis (DIGE) in visceral leishmaniasis. We identified alpha-1-acidglycoprotein and C1 inhibitor as up regulated and transthyretin, retinol binding protein and apolipoprotein A-I as down regulated proteins in visceral leishmaniasis sera in comparison with healthy controls. Western blot validation of C1 inhibitor, transthyretin and apolipoprotein A-I in a larger cohort (n = 29) confirmed significant difference in the expression levels (p Conclusions In conclusion, DIGE based proteomic analysis showed that several proteins are differentially expressed in the sera of visceral leishmaniasis. The five proteins identified here have potential, either independently or in combination, as prognostic biomarkers.</p

Mycobacterium tuberculosis polyclonal infections through treatment and recurrence.

BackgroundMixed/polyclonal infections due to different genotypes are reported in Tuberculosis. The current study was designed to understand the fate of mixed infections during the course of treatment and follow-up and its role in disease pathogenesis.MethodsSputum samples were collected on 0,1,2,3,6,12 and 24 months from 157 treatment-naïve patients, cultures subjected to Drug-Susceptibility-testing (MGIT 960), spoligotyping, MIRU-VNTR and SNP genotyping. All isolated colonies on thin layer agar (7H11) were subjected to spoligotyping.FindingsOne thirty three baseline cultures were positive (133/157, 84.7%), 43(32.3%) had mixture of genotypes. Twenty-four of these patients (55.8%) showed change in genotype while six showed different drug-susceptibility patterns while on treatment. Twenty-three (53.5%) patients with polyclonal infections showed resistance to at least one drug compared to 10/90 (11.1%) monoclonal infections (PConclusionsThe coexistence of different genotypes and change of genotypes during the same disease episode, while on treatment, confirms constancy of polyclonal infections. The composition of the mixture of genotypes and the relative predominance may be missed by culture due to its limit of detection. Polyclonal infections in TB could be a rule rather than exception and challenges the age-old dogma of reactivation/reinfection

Coupling electrochemical response of a DNA biosensor with PCR for Neisseria gonorrhoeae detection

Early diagnosis of gonococcal infections is important with regard to a patient's health and stage of infection. In this context, we report the development of an opa-gene-based electrochemical DNA biosensor for detection of Neisseria gonorrhoeae by monitoring redox peak of methylene blue indicator. The fabricated biosensor has been shown to be highly sensitive and specific when evaluated with complementary, non-complementary, and 1-base mismatch DNA sequences and polymerase chain reaction (PCR) amplified products (amplicons) of standard strain of N. gonorrhoeae (ATCC49226). The biosensor has been further evaluated using amplicons of known positive and negative clinical samples, and cut-off for positives has been determined using receiver operating characteristic curve. The sensitivity (SN), specificity (SP), positive predictive value, and negative predictive value of the biosensor have been found to be 96.2%, 88.2%, 92.6%, and 93.8%, respectively. We conclude that the combination of PCR amplification with electrochemical detection shows distinct advantage of high SN and increased SP for gonococcal detection

Genotypic, Phenotypic and Clinical Validation of GeneXpert in Extra-Pulmonary and Pulmonary Tuberculosis in India.

BACKGROUND:Newer molecular diagnostics have brought paradigm shift in early diagnosis of tuberculosis [TB]. WHO recommended use of GeneXpert MTB/RIF [Xpert] for Extra-pulmonary [EP] TB; critics have since questioned its efficiency. METHODS:The present study was designed to assess the performance of GeneXpert in 761 extra-pulmonary and 384 pulmonary specimens from patients clinically suspected of TB and compare with Phenotypic, Genotypic and Composite reference standards [CRS]. RESULTS:Comparison of GeneXpert results to CRS, demonstrated sensitivity of 100% and 90.68%, specificity of 100% and 99.62% for pulmonary and extra-pulmonary samples. On comparison with culture, sensitivity for Rifampicin [Rif] resistance detection was 87.5% and 81.82% respectively, while specificity was 100% for both pulmonary and extra-pulmonary TB. On comparison to sequencing of rpoB gene [Rif resistance determining region, RRDR], sensitivity was respectively 93.33% and 90% while specificity was 100% in both pulmonary and extra-pulmonary TB. GeneXpert assay missed 533CCG mutation in one sputum and dual mutation [517 & 519] in one pus sample, detected by sequencing. Sequencing picked dual mutation [529, 530] in a sputum sample sensitive to Rif, demonstrating, not all RRDR mutations lead to resistance. CONCLUSIONS:Current study reports observations in a patient care setting in a high burden region, from a large collection of pulmonary and extra-pulmonary samples and puts to rest questions regarding sensitivity, specificity, detection of infrequent mutations and mutations responsible for low-level Rif resistance by GeneXpert. Improvements in the assay could offer further improvement in sensitivity of detection in different patient samples; nevertheless it may be difficult to improve sensitivity of Rif resistance detection if only one gene is targeted. Assay specificity was high both for TB detection and Rif resistance detection. Despite a few misses, the assay offers major boost to early diagnosis of TB and MDR-TB, in difficult to diagnose pauci-bacillary TB

Study flow diagram.

<p>Diagnostic workflow of the patients included in the study.</p

Performance of the GeneXpert for the diagnosis of TB: Sensitivity, Specificity of GeneXpert MTB/RIF in comparison with Composite Reference Standards.

<p>Performance of the GeneXpert for the diagnosis of TB: Sensitivity, Specificity of GeneXpert MTB/RIF in comparison with Composite Reference Standards.</p

Samples with Discordant Results.

<p>Samples with Discordant Results.</p