Anil Kumar Gulati

Diagnosis of Helicobacter pylori: What should be the gold standard?

Since the discovery of Helicobacter pylori (H. pylori) in 1983, numerous detection methods for the presence of the bacterium have been developed. Each one of them has been associated with advantages and disadvantages. Noninvasive tests such as serology, (13)C urea breath test (UBT) and stool antigen tests are usually preferred by the clinicians. Serology has its own limitation especially in endemic areas while (13)C UBT is technically very demanding. The stool antigen detection method, although specific, is usually associated with poor sensitivity. The (13)C UBT is believed to be specific, but with present revelation of the fact that stomach is colonized by many other urease producing bacteria makes it questionable. Histology, culture, rapid urease test and polymerase chain reaction (PCR) are the tests which are carried out on antral biopsies collected by invasive means. Histology has been proposed to be very sensitive and specific but the question is how by simply looking the morphology of the bacteria in the microscope, one can claim that the curved bacterium is exclusively H. pylori. Rapid urease test (RUT), the doctors test, is also challenged because the presence of other urease producing bacteria in the stomach cannot be denied. Moreover, RUT has been reported with poor sensitivity specially, when density of the bacterium is low. Isolation of H. pylori is essential to investigate its growth requirements, antibiotic susceptibility testing, studying virulence factor to develop vaccine and many more explorations. It has also got several disadvantages i.e., special condition for transporting, media, incubation and few days waiting for the colonies to appear, apart from the speed essentially needed to process the specimens. Till date, majority of the microbiological laboratories in the world are not equipped and trained to isolate such fastidious bacterium. The option left is PCR methods to detect H. pyloris DNA in gastric mucosa, gastric juice, saliva, dental plaques and environmental specimens. There are speculations for false positivity due to detection of non-pylori Helicobacters due to genetic sharing; and false negativity due to low bacterial counts and presence of PCR inhibitors. However, specimen collection, transportation and processing do not require speed and special conditions. PCR based diagnosis may be considered as gold standard by designing primers extremely specific to H. pylori and targeting at least more than one conserved genes. Similarly specificity of PCR may be improved by use of internal Primers. Further, nested PCR will take care of false negatives by countering the effect of PCR inhibitors and low bacterial counts. Therefore, nested PCR based methods if performed properly, may be proposed as gold standard test.

Evaluation of Nested PCR in Diagnosis of Typhoid Fever

ABSTRACT In this study, nested PCR using H1-d primers, which is specific for Salmonella enterica serovar Typhi, was compared to blood culture and the single-tube Widal test. Results indicate that nested PCR can be used as a gold standard to determine the cutoff titer of the Widal test for diagnosis of typhoid fever.

Rapid detection of dermatophytes from skin and hair

BackgroundDermatophytes are a group of closely related keratinophilic fungi that can invade keratinized humans and animals tissues such as skin, hair and nails causing dermatophytosis. They are an important cause of superficial fungal infection.FindingsConventional methods like potassium hydroxide (KOH) microscopy and fungal culture lacks the ability to make an early and specific diagnosis. In this study we have evaluated nested Polymerase chain reaction (PCR) using primers targeting dermatophyte specific sequence of chitin synthase 1 (CHS1) gene and compared with conventional test. A total of 155 patients clinically suspected with dermatophytosis were included in the study. Of which 105 specimens were skin scrapings and 50 were hair. KOH microscopy, fungal culture and first round and nested PCR were done on clinical specimens, and results compared. Nested PCR for dermatophytes was positive in 83.8% specimens, followed by KOH microscopy (70%), first round PCR (50.8) and fungal culture (25.8).ConclusionResults indicate that nested PCR may be considered as gold standard for the diagnosis of dermatophytosis and can aid the clinician in initiating prompt and appropriate antifungal therapy.

A multiattribute utility evaluation of different methods for the detection of enteric protozoa causing diarrhea in AIDS patients.

BackgroundEnteric protozoa and sporozoa have emerged as important opportunistic parasites and can cause fatal infections in AIDS patients. The line of treatment being different for them necessitates an accurate and prompt identification of these to avoid empirical treatment. In this study which is the first of its kind from India we did a comprehensive evaluation of different techniques, comparing them on the basis of the attributes like yield, cost, time taken, expertise and infrastructure. For the first time combination of Calcoflour White and DAPI, a nuclear stain, were used to identify Microsporidia spp. Thus, a diagnostic protocol was devised for rapid, sensitive and cost effective identification of the opportunistic enteric protozoa.ResultsThe organisms isolated from the stool samples of the cases (450 HIV patients) were predominantly Cryptosporidium spp., Microsporidia spp. and Cyclospora spp. Interestingly, the control group (200 relatives of the patients who were HIV negative) showed a high incidence (21%) of Cryptosporidium spp. We found a significant increase in the sensitivity of microscopy in detecting Cryptosporidium spp. and Cyclospora spp. after formol ether concentration. Kinyouns staining was better compared to Modified safranin staining for Cryptosporidium spp. identification. Although ELISA had a sensitivity of 93.25% and specificity of 97% for Cryptosporidium spp. detection, we ranked Kinyouns staining better than ELISA because it is not affordable to most of our patients. For detecting Cyclospora cayetanensis, autoflourescence was the easiest and most cost effective method followed by Safranin technique. Combination of Calcoflour White stain and DAPI gave good results for the identification of Microsporidia spp. We assessed the above techniques and graded the attributes in the following descending order: cost effectiveness, sensitivity, ease of use and interpretation, time taken for the procedure and batch testing.ConclusionThus, we conclude that a combination of minimum three procedures should be carried out for the screening of stool specimens of HIV positive patients. Kinyouns staining should be made mandatory for every diarrheal stool sample from HIV patients. Also every laboratory should assign its own value to the attributes and apply Multiattribute utility theory or the Analytical hierarchy process to decide the most appropriate methodology.

Correlation between CD4 counts of HIV patients and enteric protozoan in different seasons – An experience of a tertiary care hospital in Varanasi (India)

BackgroundProtozoan infections are the most serious among all the superimposed infections in HIV patients and claim a number of lives every year. The line of treatment being different for diverse parasites necessitates a definitive diagnosis of the etiological agents to avoid empirical treatment. Thus, the present study has been aimed to elucidate the associations between diarrhoea and CD4 counts and to study the effect of HAART along with management of diarrhoea in HIV positive patients. This study is the first of its kind in this area where an attempt was made to correlate seasonal variation and intestinal protozoan infestations.MethodsThe study period was from January 2006 to October 2007 wherein stool samples were collected from 366 HIV positive patients with diarrhea attending the ART centre, inpatient department and ICTC of S.S. hospital, I.M.S., B.H.U., Varanasi. Simultaneously, CD4 counts were recorded to assess the status of HIV infection vis-à-vis parasitic infection. The identification of pathogens was done on the basis of direct microscopy and different staining techniques.ResultsOf the 366 patients, 112 had acute and 254 had chronic diarrhea. The percentages of intestinal protozoa detected were 78.5% in acute and 50.7% in chronic cases respectively. Immune restoration was observed in 36.6% patients after treatment on the basis of clinical observation and CD4 counts. In 39.8% of HIV positive cases Cryptosporidium spp. was detected followed by Microsporidia spp. (26.7%). The highest incidence of intestinal infection was in the rainy season. However, infection with Cyclospora spp. was at its peak in the summer. Patients with chronic diarrhea had lower CD4 cell counts. The maximum parasitic isolation was in the patients whose CD4 cell counts were below 200 cells/μl.ConclusionThere was an inverse relation between the CD4 counts and duration of diarrhea. Cryptosporidium spp. was isolated maximum among all the parasites in the HIV patients. The highest incidence of infection was seen in the rainy season.

Evaluation of Pan-Dermatophyte Nested PCR in Diagnosis of Onychomycosis

ABSTRACT In this study, nested PCR using novel primers targeting the pan-dermatophyte-specific sequence of the chitin synthase 1 gene (CHS1) was compared with KOH microscopy, culture isolation, and single-round PCR for diagnosis of 152 patients with clinically suspected onychomycosis. Results indicate that nested PCR may be considered the gold standard for the diagnosis of cases of onychomycosis for which the etiological agents are dermatophytes.

Evaluation of Nested PCR in Detection of Helicobacter pylori Targeting a Highly Conserved Gene: HSP60

Objective:  To comparatively evaluate a new nested set of primers designed for the detection of Helicobacter pylori targeting a highly conserved heat shock protein gene (Hsp60).

Prevalence of Helicobacter pylori in asymptomatic subjects—A nested PCR based study

The aim of the study was to see the prevalence of Helicobacter pylori in asymptomatic children and adults by using nested PCR which is considered to be more specific than serological methods. Saliva and stool samples of 137 healthy children (aged 8 months to 16 y) and 108 asymptomatic adults (aged 17-60 y) were collected. PCR with primers targeting Hsp60 gene sequence of H. pylori was used. H. pylori positivity with nested PCR was observed in 45.7% (112/245) of the saliva and 42.8% (105/245) of the stool specimens. Prevalence of H. pylori in saliva was found to be 2.1%, 22.7%, 55.9%, 56.0%, 68.9% and 62.9% in the age groups of < 5 y, 6-10 y, 11-16 y, 17-30 y , 31-45 y and 45-60 y, respectively. The detection rates in stool were 4.25% in < 5 y, 13.64% in 6-10 y, 50% in 11-16 y, 64% in 17-30 y, 58.62% in 31-45 y and 61.1% in 45-60 y of age groups. The most favourable age group for acquiring the infection was 11-16 y. H. pylori positivity increased with lowering of socioeconomic status. There was no gender bias in prevalence of the bacterium.

ERIC PCR and RAPD based fingerprinting of Salmonella Typhi strains isolated over a period of two decades.

Salmonella enterica serotype Typhi strains (n=113) were isolated from typhoid patients over a period of 2 decades, i.e. 1987-2006. RAPD and ERIC PCR methods were used for random whole genome typing of these strains. ERIC PCR was found to be very efficient with the discriminatory index (DI) of 0.9821 with 100% reproducibility. RAPD was satisfactory in discriminating the strains (DI=0.8978) but with poor reproducibility (40%). However, composite genotypic analysis was still better with DI of 0.9981 but with inherent poor reproducibility due to RAPD. Two major clones were observed to be circulating in the community with few unrelated strains too. The dendrogram constructed based on ERIC PCR banding pattern by involving 89 Typhi strains revealed 71 patterns, indicating that the genome of the bacterium is capable of rapid changes and variations. Thus, the spectrum of biological manifestations of human infection by S. Typhi may be related to its capacity for genetic diversity underlined by its highly plastic hypermutable genome.

Cutaneous zygomycosis: a possible postoperative complication in immunocompetent individuals.

Fungi in the class of zygomycetes usually produce serious infections in diabetics and immunocompromised hosts. Cutaneous zygomycosis is a less common form, with an unpredictable extent of anatomical involvement and clinical course. Here, we report two cases of primary cutaneous zygomycosis as postoperative complications in otherwise healthy females. Zygomycosis was suspected and specimens from the surgical debridement were examined by microbiological and histopathological studies for confirming the clinical diagnosis. Rapid diagnosis, liposomal amphotericin B, and proper debridement of affected tissue are necessary to avoid a fatal outcome.