Comparison of nested-multiplex, Taqman & SYBR Green real-time PCR in diagnosis of amoebic liver abscess in a tertiary health care institute in India.

Background & objectives: Amoebiasis is a common parasitic infection caused by Entamoeba histolytica and amoebic liver abscess (ALA) is the most common extraintestinal manifestation of amoebiasis. The aim of this study was to standardise real-time PCR assays (Taqman and SYBR Green) to detect E. histolytica from liver abscess pus and stool samples and compare its results with nested-multiplex PCR. Methods: Liver abscess pus specimens were subjected to DNA extraction. The extracted DNA samples were subjected to amplification by nested-multiplex PCR, Taqman (18S rRNA) and SYBR Green real-time PCR (16S-like rRNA assays to detect E. histolytica/E. dispar/E. moshkovskii). The amplification products were further confirmed by DNA sequence analysis. Receiver operator characteristic (ROC) curve analysis was done for nested-multiplex and SYBR Green real-time PCR and the area under the curve was calculated for evaluating the accuracy of the tests to dignose ALA. Results: In all, 17, 19 and 25 liver abscess samples were positive for E. histolytica by nested-multiplex PCR, SYBR Green and Taqman real-time PCR assays, respectively. Significant differences in detection of E. histolytica were noted in the real-time PCR assays evaluated (P<0.0001). The nested-multiplex PCR, SYBR Green real-time PCR and Taqman real-time PCR evaluated showed a positivity rate of 34, 38 and 50 per cent, respectively. Based on ROC curve analysis (considering Taqman real-time PCR as the gold standard), it was observed that SYBR Green real-time PCR was better than conventional nested-multiplex PCR for the diagnosis of ALA. Interpretation & conclusions: Taqman real-time PCR targeting the 18S rRNA had the highest positivity rate evaluated in this study. Both nested multiplex and SYBR Green real-time PCR assays utilized were evaluated to give accurate results. Real-time PCR assays can be used as the gold standard in rapid and reliable diagnosis, and appropriate management of amoebiasis, replacing the conventional molecular methods.

Polymorphic variants of β1 adrenergic receptor gene (Ser49Gly & Arg389Gly) in healthy Tamilian volunteers.

Background & objectives: Several studies reported the polymorphisms of β1-adrenergic receptor gene in healthy volunteers and its influence on cardiovascular disorders. We investigated the genotype and allele frequencies of Ser49Gly and Arg389Gly polymorphism in healthy volunteers of South Indian Tamilian population vis-à-vis other major ethnic groups. Methods: The genetic variants were determined by using Taqman 5’ nuclease assay- real time PCR analysis in 533 normal healthy volunteers (18-60 yr; M=290; F=243). The allelic discrimination analysis was done by 7700 SDS software. Results: The estimated genotype and allele frequencies of Ser49Gly and Arg389Gly polymorphism were compared with other major populations. The frequencies of the variant alleles Gly49 and Gly389 were 15.1 and 25.8 per cent respectively. Interpretation & conclusions: Our study shows that interethnic variation exists in the polymorphisms of β1-adrenergic receptor gene and the results generated in this study might serve as a genetic marker for further studies in Tamilian (South India) population.

Gly460Trp polymorphism of the ADD1 gene and essential hypertension in an Indian population: A meta-analysis on hypertension risk.

BACKGROUND: Essential hypertension is a complex genetic trait. Genetic variant of alpha adducin (ADD1) gene have been implicated as a risk factor for hypertension. Given its clinical significance, we investigated the association between ADD1 Gly460Trp gene polymorphism and essential hypertension in an Indian population. Further, a meta-analysis was carried out to estimate the risk of hypertension. METHODS: In the current study, 432 hypertensive cases and 461 healthy controls were genotyped for the Gly460Trp ADD1 gene polymorphism. Genotyping was determined by real time PCR using Taqman assay. Multiple logistic regression analysis was used to detect the association between Gly460Trp polymorphism and hypertension. RESULTS: No significant association was found in the genotype and allele distribution of Gly460Trp polymorphism with hypertension in our study. A total of 15 case-control studies were included in the meta-analysis. There was no evidence of the association of Gly460Trp polymorphism with hypertension in general or in any of the sub group. CONCLUSIONS: We found that the Gly460Trp polymorphism is not a risk factor for essential hypertension in a south Indian Tamilian population. However, the role of ADD1 polymorphism may not be excluded by a negative association study. Further, large and rigorous case-control studies that investigate gene–gene–environment interactions may generate more conclusive claims about the molecular genetics of hypertension.

Neuroparalysis and ventilatory support in severe tetanus.

In severe form of tetanus, even with maximum dose of muscle relaxants, spasms and apnoeic spells may persist and that may be life-threatening. The aim of this study was to assess the effect of neuroparalysing the patients and then providing ventilatory support in bringing about their recovery. Forty-nine adult patients of severe tetanus (Ablett's grade IIIA--6 patients and Ablett's grade IIIB--43 patients) were studied during the period from April, 1993 to February, 1996. Mean period of onset ie, period from trismus to first spasm, in these patients was 24 hours. Patients were neuroparalysed with a bolus dose of 2-4 mg of pancuronium followed by a continuous infusion of 1-2 mg/hour and simultaneously supported with mechanical ventilation until spasms subsided. Fourteen patients (28.6%) survived and rest died. Mean duration of ventilatory support on survived patients was 14.4 days. The commonest complication encountered during ventilatory support was respiratory tract infection observed in 36 patients (73.5%). Commonest cause of death was autonomic imbalance encountered in 15 patients (30.6%). Treatment of choice in severe tetanus should be neuroparalytic ventilatory support. With use of new generation ventilators and better intensive care facility, death in severe tetanus is likely to be very less.

Lymphoplasmacytic lymphoma with IgA paraproteinemia mimicking Waldenstrom's Macroglobulinemia.

Central to the diagnosis of Waldenstrom's Macroglobulinemia is the demonstration of bone marrow infiltration by lymphoplasmacytic lymphoma with Ig M monoclonal gammopathy. We describe a patient who presented with a clinical and haematological picture, highly suggestive of Waldenstrom's Macroglobulinemia, but whose serum monoclonal immunoglobulin belonged to Ig A class. Ig A secreting lymphoplasmacytic lymphoma undoubtedly exist but are exceedingly uncommon and their relationship to Waldenstrom's Macroglobulinemia needs to be clarified.

Melioidosis--a report from Pondicherry, South India.

Melioidosis is an acute infectious disease caused by a safety-pin-shaped gram-negative bacteria called Burkholderia pseudomallei. Here, we report the first case of melioidosis in a middle aged male agricultural worker, from Pondicherry. The isolation of this organism from subcutaneous nodules on the extensor aspect of his limbs underlines the diversity of its clinical presentation. Difficulty in identifying the organism which mimics any other non-fementing gram-negative bacilli (NFGNB) on cursory examination, highlights the importance of identification of NFGNB in endemic areas for specific treatment and prevention.

Human anthrax in India: urgent need for effective prevention.

Anthrax is a zoonotic illness caused by Bacillus anthracis. Sporadic cases continue to be reported from many parts of the world. From India, both sporadic cases and outbreaks are being reported regularly. The Union Territory of Pondicherry (a former French colony) lies on the coast of Bay of Bengal, where the incidence of anthrax is on the rise with 28 cases being detected in the year 1999 and 2000 alone. So far, about 34 human cases have been encountered in this region. Recently, an increase in the number of anthrax cases has been noted in veterinary and human practice in this area. Most cases have occurred in agricultural labourers who gave history of handling animal meat or skin of infected animals. The meningitic form of the disease has a very bad prognosis. Patients with this form of disease died despite treatment with high dose penicillin. The typical bacilli were seen in the CSF in all cases of anthrax meningitis and was diagnostic of the condition. The cutaneous form of illness had a benign course and responded favourably to penicillin treatment. Awareness among clinicians and mandatory reporting of cases to public health departments along with public education will help control morbidity and mortality due to anthrax. Effective immunization of animals is the other important control measure for anthrax.

A comparative study of creatinine clearance rate in type I (IDDM) and II (NIDDM) diabetic patients.

The endogenous creatinine clearance test was done in 14 Type I and 15 Type II poorly controlled diabetic patients and compared with respective age matched healthy volunteers. Type I diabetics had significantly lower creatinine clearance rate, body mass index and serum albumin levels when compared to their control group. In Type II diabetics these values remained unaltered. Both Type I and Type II diabetics had significantly higher blood sugar and glycosylated haemoglobin levels. The creatinine clearance rate had significant positive correlation with patients' body mass index and serum albumin levels. This suggests that the undernutrition of Type I diabetics may be responsible for the decreased creatinine clearance.