PCR and Genotyping for HPV in Cervical Cancer Patients.

AIMS: To devise nested multiplex polymerase chain reaction (NMPCR) protocol for detection of mucosal human papilloma viruses (HPVs) and typing of HPV-16 and -18 in formalin-fixed, paraffin-embedded (FFPE) tissues of carcinoma cervix (CaCx). SETTINGS AND DESIGN: Cross-sectional observational study. MATERIALS AND METHODS: NMPCR was done for simultaneous detection of HPV, targeting 134 bp L1 capsid gene employing GP+/mGP+ primers and typing of genotypes-16 and -18, targeting E6/E7 gene from 34 FFPE tissue blocks of CaCx and cervical intraepithelial neoplasia (CIN). Detection of 142 bp consensus sequence of L1 capsid gene was performed by nested PCR employing MY/GP+ primers. Sequencing of selected PCR amplicons of the later protocol obtained from control cell line DNA and 5 select samples were done for validation of the NMPCR protocol. STATISTICAL ANALYSIS USED: Calculation of percentage from the Microsoft Excel Software. RESULTS: Of 26 FFPE samples of CaCx, 17 (65.3%) samples were found positive for HPV by NMPCR. Amplicons of 142 bp L1 capsid gene employing MY/GP+ primers were observed in 11 (42.3%) samples of CaCx. Nearly 25% samples of CIN were positive for HPV. On sequence analysis, it was observed that the sample typed as HPV-16 by NMPCR was found to be the same on sequencing of amplicons obtained after MY/GP+ nested PCR. CONCLUSIONS: This study indicates the usefulness of our NMPCR protocol for detection of mucosal HPVs and typing of HPV-16 and -18 from FFPE tissue samples of CaCx. The NMPCR protocol may be used to detect HPV and type common genotypes-16 and -18 in fresh tissue of cervical biopsy or scrape samples for screening of CaCx.

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 doctor's 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. pylori's 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.

Nested Multiplex (NMPCR) Detection of Human Papillomavirus (HPV) 16 and 18 in Pre-invasive Lesions and its Implication in Screening of Carcinoma Cervix (CaCx).

INTRODUCTION: Carcinoma cervix (CaCx) is a preventable disease and is caused by certain high risk Papillomaviruses. In the present study, our aim was to investigate the utility of Nested Multiplex Polymerase Chain Reaction (NMPCR) in detecting Human Papillomavirus (HPV) 16 and 18 in cervical scrapes/biopsy samples and to correlate with cervical cytology/ histopathology findings. METHODS: A total of 119 females were subjected for Papanicolaou smear examination of cervical scrapes and/or histopathological examination of cervical tissues. These samples were also subjected to nested multiplex PCR targeting HPV 16/ 18 specific E6/7 gene sequences. RESULTS: HPV 16/18 were detected in 33.6% (40/119) cases included in the study. The overall HPV 16/ 18 positivity among cases with Negative for Intraepithelial Lesion or Malignancy, Low grade Squamous Intraepithelial Lesion, and High grade Squamous Intraepithelial Lesion was observed to be 20.8%, 44%, and 66.7% respectively. Positivity for HPV 16 in cases with Squamous Cell Carcinoma (SCC) was found to be 80%. HPV positivity among subjects reported with reactive cellular changes, a sub category of Negative for Intraepithelial Lesion or Malignancy, was observed to be 26.6%. CONCLUSION: HPV 16 and 18 positivity in cases reported with different stages of pre invasive lesions of CaCx, particularly in the subcategory reactive cellular changes of Negative for Intraepithelial Lesion or Malignancy, indicates that NMPCR detection of HPV 16/ 18 may be used as a screening tool for CaCx in conjunction with Papanicolaou smear examination.

Immunohistochemical Detection of p16(INK4a) in Leukoplakia and Oral Squamous Cell Carcinoma.

INTRODUCTION: Over-expression of p16(INK4a) has been reported in tissues of oral squamous cell carcinoma (SCC) associated with Human Papillomaviruses (HPVs). Immunohistochemical (IHC) detection of p16(INK4a) is an easy technique than molecular detection of HPVs, hence we investigated the presence of this protein in the most common pre-malignant and malignant oral lesions i.e. leukoplakia and SCC respectively. MATERIAL AND METHODS: We performed IHC detection of p16(INK4a) in sections of paraffin embedded formalin fixed tissues of leukoplakia with or without dysplasia (n= 21) and SCC lesions (n= 69) and correlated with various patterns of p16(INK4a) positivity with respect to histological diagnosis. RESULTS: In the present study, 71% cases of oral SCC cases were positive for p16(INK4a), of which the most common pattern was diffuse nuclear and cytoplasmic staining. Among the cases with leukoplakia, 57.1% were positive for overexpression of p16(INK4a), wherein diffuse and sporadic pattern was observed among 23.8 percent each. CONCLUSION: In the present study, significant number of oral SCC cases observed overexpressing p16(INK4a) . However HPV DNA detection based studies are needed to validate the utility of IHC detection of p16(INK4a) as a surrogate marker for HPV associated oral SCC.

Antitubercular drug resistance in four healthcare facilities in North India.

Tuberculosis (TB) is a major public-health problem in India, having the highest number of incident and multidrug-resistant (MDR) TB cases. The study was carried out to appraise the prevalence of first-line anti-TB drug resistance in Mycobacterium tuberculosis (MTB) and its patterns among different types of TB patients from different settings in a province of North India. Of 3,704 clinical specimens, 345 (9.3%) were culture-positive, and drug-susceptibility testing was carried out for 301 MTB strains. A high level of primary and acquired drug resistance of MTB was observed in the region studied, with weighted mean of 10.5% and 28.08%, 12.81% and 29.72%, 17.12% and 29.94%, 11.97% and 27.84%, and 10.74% and 23.54% for rifampicin, isoniazid, streptomycin, ethambutol-resistant and MDR cases respectively. Drug resistance was significantly higher in pulmonary (p = 0.014) and acquired drug-resistant TB cases (p < 0.001). Any drug resistance (p = 0.002) and MDR TB were significantly (p = 0.009) associated with HIV-seropositive cases. An urgent plan is needed to continuously monitor the transmission trends of drug-resistant strains, especially MDR-TB strains, in the region.

Comparison of Vi serology and nested PCR in diagnosis of chronic typhoid carriers in two different study populations in typhoid endemic area of India.

For detection of chronic typhoid carriers, nested PCR targeting flagellin the gene of Salmonella enterica subspecies enterica serotype Typhi was carried out on DNA extracted from hepatobiliary specimens from 424 autopsies which were apparently free from gallbladder pathology on postmortem examination. The second study population was 508 healthy volunteers, who did not suffer from typhoid fever during the preceding year and whose sera were subjected to detection of carriage by estimation of Vi antibody levels using an indirect hemagglutination assay. Males of both study populations had comparable rates of detection by the two methods, 6.3% by PCR and 4.1% by Vi serology. Similarly, females in both study groups had comparable frequency of detection of chronic typhoid carriage using the two methods, ie 13.1% by PCR and 15.1% by Vi serology. S. Typhi specific immunosuppression could be speculated in females of 51-60 years as only 40% were positive by Vi serology against 100% by nested PCR. Vi serology may be recommended for community based detection of chronic typhoid carriers.

Mycotic keratitis in India: a five-year retrospective study.

BACKGROUND: Mycotic keratitis is a fungal infection of the cornea. This infection is difficult to treat and it can lead to severe visual impairment or blindness. It is worldwide in distribution, but is more common in the tropics and subtropical regions. Trauma is the major predisposing factor, followed by ocular and systemic defects, prior application of corticosteroids, and prolonged use of antibiotic eye-drops. The objective of this study was to determine causative agents and to identify the predisposing factors of mycotic keratitis. METHODOLOGY: Corneal scrapings from 90 corneal ulcer patients with suspected fungal etiology were subjected to direct examination by 10% KOH mount, Gram stain and culture. RESULTS: This study included 90 subjects with corneal ulcers, based on clinical suspicion, of whom 41 cases were diagnosed with mycotic keratitis in the laboratory. Among these 41 cases, culture showed fungal growth only in 36 cases whereas the remaining five cases were positive only by potassium hydroxide (KOH) preparation. Males were more commonly affected and were mostly in the age group of 31-40 years. Aspergillus flavus was the most common fungus isolated followed by fusarium solani. CONCLUSION: Rapid diagnosis and early institution of antifungal therapy is necessary to prevent ocular morbidity and blindness. Although culture helps in definite diagnosis and identification, direct microscopic detection of fungal structures in corneal scrapes or biopsies permits a rapid presumptive diagnosis.

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.

Drug resistance pattern and clonality in H. pylori strains.

BACKGROUND: This aim of this work was to determine the in vitro activity of clarithromycin, amoxycillin, metronidazole and tetracycline against Helicobacter pylori and clonality among resistant and sensitive strains isolated from North India. METHODOLOGY: A total of 68 H. pylori isolates from peptic ulcer disease and non ulcer dyspepsia patients were examined. These strains were subjected for determination of minimum inhibitory concentration of clarithromycin, amoxycillin, metronidazole and tetracycline. For molecular characterization of resistant and sensitive strains, enterobacterial repetitive intergenic consensus sequences (ERIC) and random amplified polymorphic DNA-PCR (RAPD-PCR) methods were used. RESULTS: All the tested isolates were found resistant to metronidazole, while 65% were resistant to amoxycillin and 4.7% were resistant to clarithromycin. However, none of the isolates were found to be resistant to tetracycline. Molecular fingerprinting and cluster analysis of resistant and sensitive strains did not give clues for clonal spread of resistant strains. CONCLUSIONS: Various chromosomal mutations were seen in the putative resistance genes of resistant strains, possibly indicating selection pressure as the major cause of high resistance.

Rapid detection of dermatophytes from skin and hair.

BACKGROUND: Dermatophytes 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. FINDINGS: Conventional 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). CONCLUSION: Results 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.

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). METHODS: A total of 60 subjects having peptic ulcer diseases were tested for the detection of H. pylori using rapid urease test (RUT), histology, culture, and polymerase chain reaction (PCR) in their antral biopsy specimens. A newly designed Hsp60 gene-based primer set was evaluated against commonly used PCR primers for detection of H. pylori. RESULTS: Forty-six of the 60 study subjects were found positive for culture isolation and all the 46 culture-positive specimens were also positive with Hsp60 gene PCR. Of the 46 culture-positive specimens, 44 were positive for 16S rRNA gene, ureC gene, RUT, and histology whereas only 29 were positive with ureA gene PCR. Of the 14 culture-negative subjects, 10 were positive with 16S rRNA gene, 4 were positive with ureC (glmM) gene PCR, and 2 were positive with RUT and 1 was positive on histology. CONCLUSION: This study shows that nested amplification targeting Hsp60 gene is the most sensitive and specific with LR+ and LR- values of proportional, variant and 0, respectively, when compared with the other three PCR methods. Also, HSP60 gene-specific nested protocol was the most appropriate for detection of H. pylori in clinical specimens. This is particularly valuable because it can be used as a noninvasive method for detecting H. pylori infection in young children and also, in follow-up studies with peptic ulcer patients, on samples like feces and saliva.

Detection of Helicobacter pylori in stool specimens: comparative evaluation of nested PCR and antigen detection.

BACKGROUND: Efficacy of Helicobacter pylori stool antigen enzyme immunoassay (HpSA) and stool PCR was evaluated, before and after treatment, in a country with a high prevalence of H. pylori infection. METHODOLOGY: A total of 52 patients with dyspeptic symptoms were included in the study. Antral biopsy was collected during pre- and post-therapy periods for rapid urease test (RUT) and PCR. Similarly stool specimens for PCR and HpSA test were collected during both the periods from all 52 patients. Biopsy, PCR and RUT results together were considered the "gold standard." RESULTS: On the basis of gold standard tests, 40/52 patients were H. pylori positive. The sensitivity and specificity of HpSA test were 80% and 83.3% respectively in untreated patients. On the other hand, the sensitivity and specificity of stool PCR in untreated patients were 72.5% and 100% respectively. After eradication therapy, the results of both RUT and biopsy PCR were negative in 87.5% and positive in 12.5% of the patients. Although post treatment sensitivity of HpSA and stool PCR was equal (60%), specificity of HpSA and stool PCR were 68.6% and 97.1% respectively. CONCLUSION: The H. pylori stool tests represent a non-invasive concept for diagnosis of infection. Both HpSA and stool PCR seem to be satisfactory tests for pre-eradication as well as assessment of infection. But stool PCR is a better indicator than HpSA test in the post-eradication assessment of infection.

Association of carcinoma of the gallbladder with typhoid carriage in a typhoid endemic area using nested PCR.

BACKGROUND: Although well studied the association between chronic typhoid carrier state and carcinoma of the gallbladder (CaGB) remains unproven. METHODOLOGY: The study was performed at a tertiary care medical center in North India and involved 52 patients with CaGB, 223 patients with benign gallbladder diseases, 508 healthy individuals and, 424 corpses. For the detection of Salmonella enterica serovar Typhi, hepatobiliary specimens were subjected to DNA extraction for specific nested- PCR amplification of the S. Typhi flagellin gene. Anti-Vi S. Typhi antibodies were detected in serum samples from patients by indirect haemagglutination. RESULTS: Thirty five of the 52 (67.3%) CaGB patients were PCR-positive for the S. Typhi flagellin gene; significantly higher than for patients with benign gallbladder diseases (95/223, 42.6%; p<0.01) and corpses (35/424, 8.2%; p<0.001). The numbers of individuals that had significant anti-Vi antibody titres (> or = 160) in their serum were 20/52 (38.5%) for CaGB patients, 31/223 (13.9%) for patients with benign gallbladder diseases, and 47/508 (9.2%) for healthy individuals. CONCLUSIONS: Specific nested-PCR amplification of the S. Typhi flagellin gene in hepato-biliary specimens was more sensitive for detection of S. Typhi carriage than anti-Vi antibody titres in serum. The results demonstrate an association between typhoid carriage and gallbladder diseases, both CaGB and benign. S. Typhi specific immunosuppression is also suggested in patients with gallbladder diseases.

Evaluation of pan-dermatophyte nested PCR in diagnosis of onychomycosis.

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 diagnosis of typhoid fever.

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.