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.

Clonal diversity and drug resistance in Mycobacterium tuberculosis isolated from extra-pulmonary samples in central India--a pilot study.

In India, extrapulmonary tuberculosis (EPTB) accounts for 10 - 15% of all types of tuberculosis. To identify and compare predominant spoligotypes and drug-resistance patterns in strains of Mycobacterium tuberculosis isolated from extrapulmonary and pulmonary specimens in central India, drug susceptibility testing and spoligotyping were carried out. Spoligotyping data was analyzed using SITVIT2 database. ST11/EAI3_Ind with 33% isolates among extrapulmonary specimens and ST26/CAS1_DEL with 28% isolates among pulmonary specimens were the most predominant lineages. Multidrug resistance was found in 5.5% of the strains isolated from extrapulmonary specimens in contrast to 17% isolated from pulmonary specimens.

Rapid diagnosis of infectious diseases: the role of giant African pouched rats, dogs and honeybees.

Early detection of infection in an individual is an important strategy to prevent transmission and spread of the infection - in the community or among patients admitted in a health care facility. Over the past decade, there have been rapid advances in technology in an attempt to provide rapid, accurate tests for diagnosis of infectious diseases. With this in perspective, the use of animals' superior olfactory sensitivity to sniff out infectious diseases holds promise.

Is bleach-sedimented smear microscopy an alternative to direct microscopy under programme conditions in India?

This cross-sectional multi-centric study compared the yield of and potential benefit for detecting smear-positive pulmonary tuberculosis (PTB) by bleach sedimentation (2% sodium-hypochlorite) versus direct microscopy under programme conditions in India. Among 3168 PTB suspects, 684 (21.6%) were detected by bleach sedimentation vs. 625 (19.7%) by direct microscopy, with a proportional overall agreement of 96% (κ = 0.88). While 594 patients were smear-positive with both methods, 31 patients detected by direct microscopy were missed and an additional 90 patients were detected by bleach sedimentation. Overall, bleach sedimentation increased the yield of smear-positive TB detection; however; it also increased the time to results.

Cette étude transversale multicentrique a comparé, dans les conditions du programme en Inde, le rendement et les avantages potentiels de la détection des tuberculoses pulmonaires (TBP) à frottis positif par la sédimentation à l'eau de Javel (2% d'hypochlorite de sodium) par comparaison avec l'examen microscopique direct. Parmi 3168 sujets suspects de TBP, 684 (21,6%) ont été détectés par la sédimentation à l'eau de Javel par rapport à 625 (19,7%) par l'examen microscopique direct, avec donc une proportion de 96% de concordance globale (κ = 0,88). Alors que les résultats des frottis étaient positifs chez 594 patients par les deux méthodes, l'examen microscopique direct a raté la détection chez 31 patients et la sédimentation à l'eau de Javel a détecté 90 patients de plus. Au total, la sédimentation à l'eau de Javel a augmenté le rendement de la détection des TB à frottis positif, mais a prolongé la durée avant obtention des résultats.

En un estudio transversal multicéntrico se comparó el rendimiento y la posible utilidad de la detección de la tuberculosis (TB) con baciloscopia positiva mediante la concentración del esputo con hipoclorito de sodio (2%) y se comparó esta técnica con el examen microscópico directo, en las condiciones del programa nacional de la India. En los 3168 pacientes con presunción clínica de TB se detectaron 684 casos (21,6%) por el método de la sedimentación con lejía y 625 casos (19,7%) mediante la microscopia directa, lo cual ofrece una concordancia global del 96% (índice κ = 0,88). Quinientos noventa y cuatro pacientes presentaron baciloscopias positivas con ambas técnicas; con la técnica de sedimentación se pasaron por alto 31 de los pacientes diagnosticados por microscopia directa y la concentración con lejía permitió el diagnóstico de 90 pacientes suplementarios. En términos generales, la sedimentación con lejía aumentó el rendimiento de la detección de casos de tuberculosis con baciloscopia positiva, pero prolongó el lapso hasta la obtención de los resultados.

A pilot study to determine genetic polymorphism in Mycobacterium tuberculosis isolates in Central India.

This study was carried out to identify predominant spoligotypes responsible for transmission and prevalence of tuberculosis in central India since there is no data available about the genetic biodiversity of Mycobacterium tuberculosis isolates from patients with tuberculosis in this region. 35 strains of Mycobacterium tuberculosis were subjected to spoligotyping according to the standard protocol. A total of 25 strains out of the 35 (71.42%) could be grouped in to 6 clusters. The largest cluster comprised 8 isolates. Unique (Non-clustered) spoligotypes were seen in 10 isolates, Nine strains did not match the data base (Spol DB-4 data base). The results indicate that there may be a number of orphan strains unique to this geographical area. Further studies on a larger sample size derived from this area would help us delineate the epidemiology of Mycobacterium tuberculosis infection in this area.

Ethical issues in microbiology.

Ethical issues facing microbiologists could be considered in two parts. The first relates to the way the ethical issues during their laboratory work. The second pertains to ethical issues on the data/reports they generate for the patients or in research. In both segments, there is pressure to perform, which is exerted by both, the community, as well as peers. It has therefore become increasingly necessary to recognize the facts that unethical actions might be a frequent reality. Since some of these activities generate serious ethical concerns, both in practice and research, it is necessary for microbiologists to be aware and equipped to meet these issues in a prepared and measured way. In an attempt to highlight this requirement, this article outlines the important ethical issues and guidelines relevant to the field of Microbiology.

The incredible journey of mankind: Helicobacter pylori as the narrator.

Over the past decade, sequence differences between microbes from various geographical areas have been studied with the intent to interpret population movements of their hosts. An organism that is a reliable storehouse of such data, by virtue of its long association with its human host, is Helicobacter pylori. Functional and comparative analyses of its genome provide fascinating insights into human behaviour in the ancient past.

Diagnosis of gastrointestinal tuberculosis: Using cytomorphological, microbiological, immunological and molecular techniques - A study from Central India.

The present study included three groups: (A) age and gender matched control (n=24) with no previous signs of M. tuberculosis complex (MTBC) infection, (B) patients (n=28) diagnosed with gastro-intestinal TB (GITB), (C) patients (n=50) with clinical and histo-pathological signs of GITB, but were culture and AFB negative. Real time assay performed using fluorescence resonance energy transfer hybridization probes showed a positivity index of 36 % in group C, i.e. 18 were found reactive from the total 50 cases studied. In addition, immune characterization of these 18 cases showed depleted CD(4) (+) count and increased levels of IFN-γ and TNF-α cytokines. No positive case was found in group A, while in group B, out of total 28 cases studied 27 were found positive. A combinatorial diagnostic approach for rapid detection and characterization of GITB might provide specific therapeutic strategies for prevention and treatment of the infection in future.

Molecular epidemiology of nosocomial rotavirus infection.

The molecular epidemiology of nosocomial rotavirus infection in children admitted to the paediatric ward of a large hospital in southern India was studied by transmission electron microscopy and RNA electrophoresis on stool samples. Six hundred and twenty-six samples were collected and rotaviruses were detected in 35 of them. A predominant electropherotype was found in all the samples, and there was no seasonal variation in the incidence of infection. The predominant electropherotype pattern in this study was different from the electropherotype patterns of strains isolated elsewhere in the country and also from the pattern of the strains isolated earlier from children with acute diarrhoea at the same hospital.

Persistence of lepromatous granuloma in clinically cured cases of leprosy.

Multibacillary (MB) leprosy patients are declared cured when they become bacteriologically negative and their lesions do not show any signs of clinical activity. Whether there is also a corresponding histological clearance of skin lesions is not known. The present study was carried out on the histopathology of the skin of 40 MB cases who were declared cured with multidrug (MDT). In 22 of them, small lepromatous granulomas were seen to persist, although acidfast bacilli were not found in them. The presence of these granulomas was not found to have any significant associated with the duration of the disease, length of treatment, or the period of cessation of therapy.

Role of antineural antibodies in perpetuation of a pre-existing peripheral nerve damage in leprosy.

This study was carried out in order to find out whether antineural antibodies had a role to play in perpetuating pre-existing nerve damage in leprosy. Indirect ELISA was carried out on sera from 20 leprosy patients and five normal controls using antigen prepared from peripheral nerves of a cured bacteriologically negative leprosy patient. None of the patients had significant levels of IgG antibodies whereas eight of them (40%) had significant levels of IgM antibodies. However, there was no correlation with duration of disease, treatment received, nerve enlargement or active neuritis. The nature of these antibodies is discussed.

The role of antiperipheral nerve antibodies in nerve damage in leprosy.

The objective of this study was to determine the role of antineural antibodies in leprosy. Indirect ELISA using antigen prepared from normal human peripheral nerves was carried out on the sera from 100 leprosy patients and 18 normal controls. In total, 9% of the patients had demonstrable levels of IgG antineural antibodies and 11% had demonstrable levels of IgM antibodies. There was no correlation with the type of leprosy, bacteriological index, treatment taken, the presence of a reactional state, the presence of enlarged nerves or active neuritis.