Evaluation of highly conserved hsp65-specific nested PCR primers for diagnosing Mycobacterium tuberculosis.

OBJECTIVE: To evaluate the sensitivity and specificity of a new nested set of primers designed for the detection of Mycobacterium tuberculosis complex targeting a highly conserved heat shock protein gene (hsp65). DESIGN: The nested primers were designed using multiple sequence alignment assuming the nucleotide sequence of the M. tuberculosis H37Rv hsp65 genome as base. Multidrug-resistant Mycobacterium species along with other non-mycobacterial and fungal species were included to evaluate the specificity of M. tuberculosis hsp65 gene-specific primers. RESULTS: The sensitivity of the primers was determined using serial 10-fold dilutions, and was 100% as shown by the bands in the case of M. tuberculosis complex. None of the other non M. tuberculosis complex bacterial and fungal species yielded any band on nested polymerase chain reaction (PCR). The first round of amplification could amplify 0.3 ng of the template DNA, while nested PCR could detect 0.3 pg. CONCLUSION: The present hsp65-specific primers have been observed to be sensitive, specific and cost-effective, without requiring interpretation of biochemical tests, real-time PCR, sequencing or high-performance liquid chromatography. These primer sets do not have the drawbacks associated with those protocols that target insertion sequence 6110, 16S rDNA, rpoB, recA and MPT 64.

Antiphospholipid antibodies in young Indian patients with stroke.

BACKGROUND: Stroke may be caused by antiphospholipid antibodies (APL), especially in young persons without other risk factors. AIM: The aim of this study was to determine the prevalence of two clinically significant APL-anticardiolipin antibody (ACL) and lupus anticoagulants (LA) in young patients presenting with sudden neurological deficit. SETTINGS AND DESIGN: A case-control study performed in a large tertiary care government hospital. MATERIALS AND METHODS: Ten milliliters of blood was collected from 51 consecutive young patients (age less than 45 years) diagnosed as ischemic stroke and 50 healthy age- and sex-matched controls. STATISTICAL ANALYSIS: Statistical analysis was done using Epi Info (TM) 6 software. RESULTS: Overall, the risk factor profile was: Smoking (23.5%), positive family history (13.7%), hyperlipidemia (8%), and diabetes mellitus (3.6%). APL (LA and ACL) were present in 29.4% of the samples and in 4% of controls. The 'P' value for ACL and LA was 0.03 and 0.02 respectively, but the maximum level of ACL was 25 GPL units only. One-fourth of the patients were smokers and one-sixth had a family history of thrombosis ( P = 0.048 and 0.036, respectively). CONCLUSIONS: APL, positive family history and smoking were significantly associated with stroke in the young. We advocate screening for APL in all young patients with stroke.

Immunochromatographic Methods in Malaria Diagnosis.

BACKGROUND: Malaria remains a major cause of morbidity and mortality in India. This study was carried out to evaluate the use of parasite lactate dehydrogenase (pLDH) test in diagnosis of malaria. METHODS: Blood slides of 400 patients who presented with fever including 104 patients with clinical features suggestive of malaria were studied. The results were compared with microscopy and another immunochromatography test (ICT) based on detection of histidine rich protein-2 antigen [Pfhrp-2] secreted by Plasmodium falciparum. RESULT: In this study the sensitivity and specificity for detection of Plasmodium vivax was 100% while for Plasmodium falciparum the values were 96% and 100% respectively. CONCLUSION: ICT is useful for diagnosis of malaria caused by Plasmodium falciparum in field but microscopy of a well-prepared blood smear must not be omitted in a laboratory setting.