A novel ready-to-use dry-reagent polymerase chain reaction for detection of Escherichia coli & Shigella species.

Background & objectives: Polymerase chain reaction (PCR) has wide acceptance for rapid identification of pathogens and also for diagnosis of infectious conditions. However, because of economic and expertise constraints, a majority of small or peripheral laboratories do not use PCR. The objective of the present study was to develop a dry-reagent PCR assay as an alternative to conventional PCR to assess its applicability in routine laboratory practice using malB gene for identification of Escherichia coli as a model. Methods: A total of 184 isolates were selected for the study comprising clinical isolates of E. coli and non-E. coli including Shigella sp. and a few other control strains. The DNA was isolated from all the isolates. The isolated DNA as well as the overnight grown bacterial cultures were subjected to both conventional wet PCR and dry-reagent PCR. Results: The genomic DNA isolated from E. coli showed amplification of malB gene in both conventional wet and dry-reagent PCR and the band was observed at 491 bp. In dry-reagent PCR, the overnight grown E. coli cells also showed positive result. The non-E. coli strains other than Shigella sp. showed negative in both conventional wet and dry-reagent PCR. Shigella sp. showed positive in both conventional wet and dry-reagent PCR. Interpretation & conclusions: Considering the elimination of genomic DNA isolation step, and similar results with the conventional wet PCR, dry-reagent PCR may be a good alternative for the conventional wet PCR.

Development of a dry-reagent mix-based polymerase chain reaction as a novel tool for the identification of Acinetobacter species and its comparison with conventional polymerase chain reaction.

BACKGROUND: Nosocomial infections are often caused by multidrug-resistant bacteria and the incidence is increasing. Acinetobacter, a Gram-negative bacillus, is commonly associated with the use of intravascular catheterization and airway intubation. Polymerase chain reaction (PCR) for identification of Acinetobacter baumannii from samples has been standardized that use conventional wet-reagent mix. We have designed and optimized a dry-reagent mix for identification of Acinetobacter species by PCR. The dry-reagent mix can be stored at room temperature, has less chances of contamination, and thus can be used at point-of-care diagnosis. AIM AND OBJECTIVE: The present work was focused on comparing the sensitivity and specificity of dry-reagent PCR mix over conventional wet-reagent PCR mix for identification of Acinetobacter species. MATERIALS AND METHODS: Conventional wet-reagent mix based and dry-reagent mix based PCR were carried out for the DNA isolated from Acinetobacter species. The latter was also applied directly on bacterial growth without prior DNA extraction process. Equal numbers of bacterial isolates other than Acinetobacter species were also subjected to identification by the same protocols for determining the sensitivity and specificity of the test. RESULTS: The Acinetobacter species showed amplification of the target rpoB gene and the band was observed at 397 bp. The dry-reagent PCR mix results matched completely with the conventional wet-reagent PCR mix assay. All the non-Acinetobacter isolates were negative for the PCR. This indicates that the test is highly specific. The dry-reagent mix also contained an enzyme resistant to PCR inhibitors and capable of amplifying DNA directly from cells. CONCLUSION: Performance of dry-reagent PCR mix without the need for DNA extraction and preparation of a PCR mix proved to be more sensitive and reduce the handling error, minimizes the time, manual work, and skilled labor.

Global eradication of measles: Are we poised?

Measles, a highly infectious viral disease is the next target for eradication following poliovirus. Decades of experience with highly effective vaccination has invigorated us to take on this virus. The task is not only Titanic but is laced with intricate issues. Recently, an outbreak of fever with rash occurred on a tertiary care teaching hospital campus and was confirmed serologically as measles outbreak by IgMELISA. Therefore, we searched the literature related to outbreaks, transmission of the measles virus, age groups involved, vaccination strategies, vaccination failure and epidemiological features of the disease and reviewed the possible reasons for such outbreaks and problems in the global eradication of the virus.

Trend of human brucellosis over a decade at tertiary care centre in North Karnataka.

BACKGROUND: Brucellosis is an important zoonotic disease. India having a major agrarian population is expected to have a higher prevalence. However, due to lack of laboratory facility or awareness among clinicians, the disease is largely underreported. The aim of this study was to know the prevalence and trend of human brucellosis over a decade, in patients attending a teaching hospital in North Karnataka, and to understand their geographical distribution. MATERIALS AND METHODS: The study was conducted from January 2006 to December 2015 at a tertiary care teaching hospital in North Karnataka. A total of 3610 serum samples were evaluated from suspected cases of brucellosis. All serum samples were initially screened by Rose Bengal plate test, and positive samples were further analysed by Serum agglutination test (SAT) using standard Brucella abortus antigen from Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India. A titre above or equal to 1:80 IU/ml was considered as positive. Demographic data such as age, sex and native place of these patients were also analysed. RESULTS: We observed that human brucellosis is present in North Karnataka. The overall seropositivity of brucellosis in suspected cases was 5.1%. The positive titres ranged from 1:80 to 163,840 IU/ml. The majority of the patients were from Gadag, Koppal and Haveri districts of North Karnataka. CONCLUSION: Our study confirms the presence of human brucellosis in the northern part of Karnataka. Further studies to understand the prevalence of animal brucellosis in these areas will help in implementing prevention measures.

Antibacterial drug resistance and its impact on dentistry.

The continuing emergence of antimicrobial resistant bacteria is a global health problem. Multi-drug resistance is now widespread. Resistance rates differ noticeably on a worldwide, regional and even institutional basis. Because antibiotics are commonly used in dentistry, the dental community is not spared from this threat of microbial resistance to antibiotics. This article presents an overview of antimicrobial drug resistance, discusses how this large and expensive problem affects the dental community and what we can do to change the situation, both as concerned citizens and as dental practitioners.

Association of platelet count and serological markers of dengue infection- importance of NS1 antigen.

INTRODUCTION: Dengue is an acute viral infection with potential fatal complications. Specific antibody detection has been the mainstay of diagnosis which is prone for both false positive and false negative reactions. The newer parameter NS1 appears to be highly specific and reliable for diagnosis of dengue infection from the first day of fever. Platelet count is the only accessory test for diagnosis of dengue infection in the peripheral laboratories. Therefore, we tried to evaluate the association of platelet counts against NS1 and IgM/IgG in dengue infections. MATERIALS AND METHODS: Serum samples from clinically suspected dengue cases were tested for NS1, IgM and IgG by immunochromatography-based test. Platelet counts were obtained for all positive cases and 150 dengue seronegative cases of fever that served as controls. Test results of dengue-specific parameters were compared against platelet counts. The proportions obtained were compared by Standard error of the difference between the proportions (SEP test). RESULTS: Of 2104 samples tested, 320 were positive for one or more dengue parameters. Of the 320, 95 were positive for NS1 only, 161 showed IgM only while 9 showed IgG only. More than one marker was detected in the remaining 55 samples. Thrombocytopenia was more consistently associated whenever NS1 was detected compared to antibody detection (P value <0.001). CONCLUSIONS: Inclusion of NS1 in the diagnosis of dengue increases the detection rate significantly. In cases of fever, thrombocytopenia is more consistently found in dengue positive rather than dengue negative subjects. It correlates well when NS1 and IgM are detected simultaneously.

Comparative study of adherence of oral Candida albicans isolates from HIV sero-positive individuals and HIV sero-negative individuals to human buccal epithelial cells.

AIM: Candida albicans occurs as a commensal of the gastrointestinal tract. Under predisposing conditions, candida can produce a broad array of infections. HIV seropositive individuals show increased oral colonization compared to the HIV seronegative healthy individuals. C. albicans shows a variety of pathogenic factors. We have studied one such factor here; the adherence property of C. albicans isolated from HIV seropositive individuals and HIV seronegative to Human Buccal Epithelial Cells (HBEC) of normal healthy individuals. MATERIALS AND METHODS: Concentrated oral rinse specimen were collected from 50 healthy volunteers (control group) and 25 HIV positive individuals (test group) and used for isolation of C. albicans. Adherence assay was done using C. albicans isolates from both groups on HBEC collected from HIV sero-negative, normal individuals. The adherence assay method described by Kimura and Pearsall was used with minor modification. STATISTICAL ANALYSIS USED: The results of Adhesion assay were subjected to statistical analysis using student "t" test. RESULTS: C. albicans isolated from both the groups were tested for their adherence property to normal HBEC. The isolates from test group showed more adherence to HBEC compared to those of the control group, with average rate of adherence being 56.6%. The control group showed average adherence rate of 29.1%. This was statistically significant with p value equal to 0.05. CONCLUSION: C. albicans from HIV infected individuals showed significant rise in degree of adhesion to the buccal epithelial cells than the isolates from healthy controls, suggesting the enhancement of virulence factors such as adherence in the presence of predisposing condition.

Isolation of Salmonella enterica serotype Isangi from a suspected case of enteric encephalopathy.

Nontyphoidal salmonella species are thought to be potentially infectious to humans and many are documented to cause human diseases. We isolated S. Isangi from the blood of a 30-year-old man with complaints of diarrhoea, fever, and altered sensorium. The serotype of the isolate was confirmed at National Salmonella Centre (Vet.), Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izzatnagar, India. The isolate was not an extended spectrum beta-lactamase (ESBL) producer and the patient responded well to ceftriaxone. We reviewed the literature concerning infections caused by salmonella; however, did not find any report related to S. Isangi infection in human beings from India.

Phenotypic detection of inducible clindamycin resistance among Staphylococcus aureus isolates by using the lower limit of recommended inter-disk distance.

CONTEXT: Clindamycin is one of the important alternative antibiotics in the therapy of Staphylococcus aureus, particularly in methicillin-resistant S. aureus (MRSA) infections. Inducible clindamycin resistance (iMLS B--inducible Macrolide-Lincosamide-Streptogramin B resistance) is a critical factor in antimicrobial susceptibility testing. AIMS: To know the rate of inducible clindamycin resistance among clinical isolates of Staphylococcus aureus in our hospital by Disk approximation test (D-test) using the average recommended inter-disk distance and comparing the results with that of D-test using the lower limit of recommended inter-disk distance. MATERIALS AND METHODS: A total of 51 erythromycin-resistant and clindamycin-susceptible S. aureus isolates were subjected to disk approximation testing with 21 +/- 1 mm and 15 mm edge-to-edge distance between the clindamycin and erythromycin disks. STATISTICAL METHODS: Z-test levels. RESULTS: Among 51 erythromycin-resistant and clindamycin-susceptible S. aureus isolates, 25 (49%) were recorded as inducible clindamycin resistant by D-test with 21 +/- 1 mm edge-to-edge distance between the clindamycin and erythromycin disks. When we re-tested all the 51 strains by D-test with 15 mm inter-disk distance, we identified 14% more iMLS B strains previously reported as D-test negative. Z-test for MRSA indicates that 15 mm edge-to-edge distance has significant advantage. CONCLUSIONS: Since the incidence of inducible clindamycin resistance is high (63% in our study), accurate identification of inducible clindamycin resistance is important to prevent therapeutic failure in infections caused by these strains. We suggest the use of D-test with 15 mm edge-to-edge inter-disk distance for detecting iMLS B .