Detection of parvovirus B19 in selected high-risk patient groups & their phylogenetic & selection analysis

Background & objectives: Human parvovirus B19V (B19V) is known to be associated with erythema infectiosum commonly in children, aplastic crisis, especially in persons with underlying haemolytic disorders, hydrops fetalis in pregnancies and arthritis. This cross-sectional study was aimed to determine the presence of B19V infection in childhood febrile illnesses, association of B19V with arthropathies and in adult patients with end-stage renal disease (ESRD) on dialysis. The genetic diversity among the sequences was also analysed. Methods: A nested polymerase chain reaction (nPCR) assay was used for B19V DNA targeting VP1/VP2 region and used for testing 618 patients and 100 healthy controls. Phylogenetic analysis on nucleotide and amino acid sequences was carried out to compare our sequences with other Indian strains and global strains. Results: Among 618 samples tested, seven (1.13%) were found positive. The phylogenetic analysis revealed that all the seven sequences belonged to genotype 1 and showed low genetic diversity. The clustering pattern of seven sequences was similar both by nucleotide and by predicted amino acid sequences. The fixed effects likelihood analysis showed no positive or negatively selected sites. Interpretation & conclusions: Seven samples (4 from non-traumatic arthropathies, 2 from patients with ESRD and 1 from febrile illness patient) were found positive by nPCR. When our seven sequences were compared with global strains, the closest neighbour was other Indian strains followed by the Tunisian strains.

Frequency of extended spectrum β-lactamase producing urinary isolates of Gram-negative bacilli among patients seen in a multispecialty hospital in Vellore district, India.

Extended‑spectrum beta‑lactamase (ESBL) producing strains of Coliform bacilli are on the rise and present a major threat especially in India. We assessed the frequency of ESBL producers among urinary isolates from patients presenting urinary tract infections. ESBL screening was done using Double Disk Synergy Test (DDST) and confirmed using E‑test and Polymerase Chain Reaction (PCR). With E‑test, 92.2% were positive for ESBL. In PCR, 100% strains were positive for any of the three gene targets tested. CTX‑M was positive in majority of the strains followed by TEM and SHV. Two (3.22%) strains were positive for all the three genes; 21% strains were positive for both TEM and CTX‑M genes. There was no statistically significant difference in the findings of E‑test and PCR testing in the determination of ESBL producers (Fisher exact test P = 0.15). The strength of agreement between them was ‘fair’ (k = 0.252). Continuous monitoring of ESBL producers among Indian strains is important to rationalize the antibiotic policy to be followed.

Molecular characterization of CTX-M type Extended Spectrum Beta Lactamase producing E. coli isolated from humans and the environment.

Purpose: Escherichia coli is a common pathogen causing community- and hospital-acquired infections. The infections caused by the Extended Spectrum Beta Lactamase (ESBL) enzymes-producing E. coli hinder antibiotic treatment. Materials and Methods: Plasmid DNA samples were subjected to PCR specifi c for TEM, SHV and CTX-M genes obtained from 110 E. coli strains isolated from hospitalized patients, healthy individuals and environment in Vellore, South India. Results: Among the 110 isolates tested, 21.8% were positive for TEM and 2.7% positive for SHV and 91.8% positive for CTX-M. The proportion of CTX-M positive E. coli was not statistically different between the study groups. Nineteen of 20 strains were CTX-M-15 type and the other was CTX-M-14 type. The phylogenetic analysis of 19 strains clustered with the pandemic CTX-M-15-ST131 strain, indicating this as an evolving global problem for antibiotic therapy. The geomapping of samples indicated ‘hotspot’ areas of healthy individuals, patients and the environmental samples. Conclusion: The spatial presentation of GIS mapping allowed identifi cation of clustering among patients and healthy individuals and contaminated environmental points.

Incidence of extended spectrum beta lactamase producing Escherichia coli among patients, healthy individuals and in the environment.

We investigated the faecal carriage of extended spectrum β‑lactamases (ESBL) producing Escherichia coli in different groups of human subjects and in the environment. A total of 363 E. coli strains were isolated from stool samples of patients (n = 77), healthy subjects (n = 170) and from different environmental samples (n = 116). A total of 124 ESBL producing E. coli strains were isolated in this study. The frequency of ESBL producing E. coli was found to be highest (60.3%) among the strains isolated from patients, followed by healthy individuals (38%) and the environment (10.5%). The environment was observed to have a very low number of ESBL producing E. coli.

Could the products of Indian medicinal plants be the next alternative for the treatment of infections.

Indian medicinal plants are now recognized to have great potential for preparing clinically useful drugs that could even be used by allopathic physicians. Traditionally, practitioners of Indian medicine have used plant products in powder, syrup or lotion forms, without identification, quantification and dose regulation, unlike their allopathic counterparts. The present review explores the immense potential of the demonstrated effect of Indian medicinal plants on microbes, viruses and parasites. In the present context, with the available talent in the country like pharmaceutical chemists, microbiologists, biotechnologists and interested allopathic physicians, significant national effort towards identification of an "active principle" of Indian medicinal plants to treat human and animal infections should be a priority.

Comparative evaluation of two polymerase chain reactions targeting different genomic regions to detect Mycobacterium tuberculosis in sputum.

Purpose: Tuberculosis remains an important health problem all over the world, especially in resource poor settings like India. The Ziehl-Neelsen (ZN) staining of sputum smear is still the method of choice in the diagnosis of tuberculosis in spite of its low sensitivity and specificity. This paper evaluates comparison of two different polymerase chain reaction (PCR) assays with sputum smear findings to detect Mycobacterium tuberculosis. Materials and Methods: A total of 191 sputum samples were collected from 84 patients attending a tertiary care hospital, who were suspected of having pulmonary tuberculosis, were examined by PCR targeting two different genomic regions, namely, TRC 4 by non-nested format and IS6110 insertion element by nested format in comparison to ZN staining of sputum smears. Results: Among the patients tested, 20.24% (Mid-p 95%CI: 31.5-52.4) were smear positive, 7.14% (Mid-p 95%CI: 2.94-14.26) were positive by TRC 4 PCR and 41.67% (Mid-p 95%CI: 12.7-29.8) were positive by IS6110 nested PCR (nPCR). The median age of overall positive cases was 42 years. Among the nPCR positives, the median for age of rural and peri-urban community was 46 and 32 years, respectively. The kappa coefficient between smear findings and TRC4 PCR findings was 0.27 and an agreement of 0.83 was observed (Z = 2.99; one-tailed P = 0.001). TRC 4 PCR picked two unique positives that were negative by smear and IS6110 nPCR. Conclusion: The non-nested TRC 4 PCR showed inability for accurate detection of M. tuberculosis in sputum samples. The study concluded that the nPCR targeting IS6110 is superior and more sensitive than TRC4 PCR.

Evaluation of a nested PCR targeting IS6110 of Mycobacterium tuberculosis for detection of the organism in the leukocyte fraction of blood samples.

Purpose: Tuberculosis poses a serious health problem in resource-poor settings such as India. Polymerase chain reaction (PCR) is presently seen as a promising alternative to conventional smear microscopy and culture techniques. Undiagnosed fever is a condition where the aetiology could include tuberculosis in a significant percentage. This paper evaluates a nested PCR (nPCR) using Hotstar Taq for the detection of M. tuberculosis in patients with febrile illness using insertion element, IS6110 as a target. Material and Methods: A total of 355 samples (301 HIV status unknown and 54 HIV seropositives) from patients primarily with febrile illness were tested for the presence of M. tuberculosis. Blood culture was done in a commercial automated blood culture system and nPCR in DNA extracts from buffy coat samples. Hotstar Taq polymerase was used to enhance the sensitivity of nPCR and the lower limit of detection was determined by using cloned plasmid. Results: Among the patients tested, 2% were positive by automated culture system and 6.8% of patients were positive by nPCR. Majority of the positives were from HIV seropositive individuals. The sensitivity of the nPCR was 100% and the specificity was 95.1%. The lower limit of detection was less than 1 genome copy per microlitre. Among the nPCR positives, patients from rural community were significantly higher than from the peri-urban community. Conclusions: The nPCR had a high sensitivity and specificity on buffy coat samples using Hotstar Taq polymerase in the reaction mix. Thus the technique is a valuable tool in the diagnosis of tuberculosis.