Amplification of the 3q chromosomal region as a specific marker in cervical cancer.

OBJECTIVE: Chromosome 3q gain has been consistently observed in cervical intraepithelial neoplasia grades 2 and 3 (CIN 2,3) and squamous cell carcinomas of the cervix. There are a number of potential clinical uses of testing for 3q gain in liquid cytology specimens, including the identification of subsets of women with atypical squamous cells of undetermined significance or low-grade squamous intraepithelial lesion cytology who are at greatest risk of having CIN 2,3 and would thus benefit most from immediate colposcopy. The objective of this study was to establish the sensitivity and specificity of 3q gain for discriminating between CIN 2,3 and normal. STUDY DESIGN: Residual cytology specimens were collected from 199 women. Liquid-based cytology (LBC) was used for the selection of subjects, with women with high-grade squamous intraepithelial lesion or high-grade squamous intraepithelial lesion who had colposcopy and adjudicated biopsy-confirmed CIN 2,3 forming the disease-positive group (n = 28) and women doubly negative for both cytology and high-risk human papillomavirus (hrHPV) testing forming the disease-negative group (n = 171). A single slide was prepared from each residual LBC specimen and analyzed for 3q gain by fluorescent in situ hybridization, using a probe specific for the 3q26 region and a control probe for the chromosome 7 centromere. Two approaches were compared for the determination of 3q gain. The first was based on the analysis of an entire cervical cytology slide for the presence of rare cells with a high copy number (>4 copies) for the 3q locus. The second approach was based on the analysis of 400 cells to determine the percentage with 3 or more copies of the 3q locus. RESULTS: Using the approach based on the detection of rare cells with a high copy number (>4 copies) for the 3q locus, 26 of the specimens from women with CIN 2,3 and none of the 171 specimens from women who were both hrHPV and cytology negative was positive for 3q gain. This translates to a sensitivity of 92.9% (95% confidence interval [CI], 76.5-98.9%), a specificity of 100% (95% CI, 97.8-100%), a positive predictive value of 100% (95% CI, 86.7-100%), and a negative predictive value of 98.8% (95% CI, 95.9-99.8), for distinguishing CIN 2,3 from normal. CONCLUSION: These data support the potential clinical use of 3q gain for the evaluation of women in a number of clinical situations, including women with atypical squamous cells of undetermined significance, low-grade squamous intraepithelial lesion, and those who are hrHPV positive.

Is there a gain in chromosome 3q in the pathway to anal cancer?

BACKGROUND: Chromosome 3q gain has been identified in human papillomavirus-infected cervical cancer cells. OBJECTIVE: We sought to identify the presence of chromosomal 3q gain in anal neoplasia. DESIGN: This was a retrospective cohort. SETTINGS: The study was conducted in a group colorectal surgery practice. PATIENTS: Fifty-two patients with no dysplasia, low-grade dysplasia, high-grade dysplasia, or anal cancer were studied. INTERVENTIONS: Pairs of biopsy specimens were paraffin embedded and reviewed. One of each slide pair was stained with hematoxylin and eosin and the second processed for fluorescence in situ hybridization. The hybridized set was deparaffinized first and then hybridized with a probe for the chromosome 3q26 region. Then, slides were scanned using an automated fluorescence microscopy system that analyzed defined areas of the tissue to enumerate all of the nuclei for hybridized probe signals to detect chromosome 3q gain. MAIN OUTCOME MEASURES: We measured for gain in chromosome 3q26. RESULTS: We identified chromosome 3q gain in 7 (78%) of 9 patients with squamous-cell cancer, 8 (53%) of 15 high-grade dysplasia samples, 0 of 12 low-grade dysplasia samples, and 0 of 16 samples with no dysplasia. The sensitivity for high-grade or invasive neoplasia was 58%, with a specificity of 100%. The positive predictive value of the test was 100% for detecting high-grade dysplasia and/or squamous-cell cancer from no dysplasia, and the negative predictive value of the test was 62%. LIMITATIONS: This study was limited by its small sample size and retrospective design. CONCLUSIONS: Chromosome 3q gain represents an important shared pathway to tumorigenesis in cervical and anal neoplasia. Multiple potential diagnostic roles exist for this easily performed test in the evaluation of anal neoplasia.

Childhood Intra-Thoracic Tuberculosis Clinical Presentation Determines Yield of Laboratory Diagnostic Assays.

Diagnosis of intra-thoracic tuberculosis (ITTB) in children is difficult due to the paucibacillary nature of the disease, the challenge in collecting appropriate specimens, and the low sensitivity of smear microscopy and culture. Culture and Xpert MTB/RIF provide higher diagnostic yield in presumptive TB in adults than in children. Current study was designed to understand poor yield of diagnostic assays in children. Children with presumptive ITTB were subjected to gastric aspirates and induced sputum twice. Samples were tested by Ziehl-Neelsen stain, Xpert MTB/RIF-assay, and MGIT-960 culture. Subjects were grouped as Confirmed, Unconfirmed, and Unlikely TB, and classified as progressive primary disease (PPD, lung parenchymal lesion), and primary pulmonary complex (PPC, hilar lymphadenopathy) on chest X-ray. Of children with culture-positive TB 51/394 (12.9%), culture-negative TB 305 (77.4%), and unlikely TB 38 (9.6%), 9 (2.3%) were smear positive, while 95 (24.1%) were Xpert-MTB/RIF positive. Xpert-MTB/RIF detected 40/51 culture confirmed cases (sensitivity 78.4% and NPV 96.3%). Culture was positive in more children presenting as PPD (p < 0.04). In culture-negative TB group, Xpert positivity was seen in 31% of those with PPD and 11.9% in those with PPC (p < 0.001). Conclusion: Xpert-MTB/RIF improved diagnosis by 2-fold and increased detection of MDR-TB. Both liquid culture and Xpert-MTB/RIF gave higher yield in children with lung parenchymal lesions. Children with hilar lymphadenopathy without active lung parenchymal lesions had poor diagnostic yield even with sensitive nucleic acid amplification tests, due to paucibacillary/localized disease, suggesting possible utility of invasively collected samples in early diagnosis and treatment.

Granulomatous hepatitis by Nocardia species: An unusual case.

A case of granulomatous hepatitis due to Nocardia is reported here. The case patient was a 63-year-old immunocompetent man who presented with persistent fever, weight loss, and malaise. Radiology suggested an enlarged liver with dense diffuse to multiple tiny micronodular areas of parenchymal involvement, possibly granulomatous. Liver biopsy showed necrotizing granulomas and anti-tuberculosis therapy was initiated, but the patient showed no improvement. A repeat liver biopsy showed similar histopathology; however PCR for Mycobacterium tuberculosis was negative, while MGIT 960 culture grew filamentous Gram-positive bacilli, acid-fast by 1% H2SO4, identified biochemically as Nocardia spp. 16S rRNA sequencing confirmed Nocardia spp. A diagnosis of granulomatous hepatitis due to Nocardia spp. was made. Treatment based on drug sensitivity testing was initiated, resulting in a resolution of symptoms. The patient's history revealed that stray dogs adopted by his family had skin lesions, likely canine distemper (two newborn puppies had died recently). Nocardia is known to co-infect animals with distemper. This could have been the possible source of a zoonotic infection to the case patient. Nocardia spp. are seldom reported from sites other than the lungs, skin, or brain; the current case highlights the involvement of the liver. Due to the granulomatous tissue response, it could represent a differential diagnosis of tuberculosis in such cases.

Earlier and higher rates of cytomegalovirus infection in pediatric liver transplant recipients as compared to adults: An observational study.

AIM: To study and compare the incidence and time of occurrence of cytomegalovirus (CMV) infection in the posttransplant period in adult and pediatric liver transplant recipients. MATERIALS AND METHODS: Consecutive live donor liver transplant recipients not on CMV prophylaxis, were prospectively enrolled from March 2012 to September 2015 and followed up for 1 year post transplant to look for CMV infection. For analysis, patients were divided into pediatric (up to 18 years) and adult (>18 years) age groups. RESULTS: The study population of 146 patients consisted of 132 adult and 14 pediatric patients. Overall CMV infection posttransplant was seen in 54/146 (36.98%) patients, and 16/54 (29.6%) patients developed CMV disease. Post-transplant CMV infection rate was significantly higher in pediatric patients(10/14 [71.4%]) as compared to adults (44/132 [33.4%]) (P = 0.004). Among adults, CMV infection was seen in 22 (50%) patients in the 1st month, 13 (29.5%) patients in the 2nd month, 5 (11.4%) patients in the 3rd month, 2 (4.5%) patients in the 4th month, and 1 (2.3%) patient each in the 5th and 6th month. However, in pediatric patients, all the patients having CMV infection had it in the 1st-month posttransplant (P = 0.003). The median time of occurrence of CMV infection was 11.5 (7.75-19.00) days in pediatric patients versus 30 (18.5-54.5) days in adult patients (P = 0.001). CONCLUSIONS: The results of this study show a clear difference in the incidence and timeline of posttransplant CMV infection in pediatric patients as compared to adults.

Predictors of Microbiologically Confirmed Intrathoracic Tuberculosis.

OBJECTIVE: To identify risk factors for microbiologically confirmed intrathoracic tuberculosis in children. METHODS: Children, 6 mo to 15 y of age, attending the out-patient department of a tertiary care centre in India, with probable intrathoracic tuberculosis were enrolled. Microbiological confirmation of tuberculosis was defined as positivity on smear (Ziehl-Neelsen staining) and/or Xpert MTB/RIF and/or MGIT-960 culture. Association of various factors with microbiological confirmation were assessed by univariate and multivariate analysis. RESULTS: Microbiologic confirmation was documented in 39 (25%) of 153 patients enrolled. On univariate analysis, microbiological positivity was associated with female gender, higher mean (SD) age [136.6 (31.8) vs. 117.3 (41.4) mo], parenchymal lesion on chest radiograph, low body mass index for age, having symptoms of cough and weight loss, lower mean (SD) hemoglobin [10.4 (1.37) g/dl vs. 11(1.52) g/dl; p = 0.04], and higher mean (SD) monocyte: lymphocyte ratio [0.38 (0.30) vs. 0.24 (0.02); p = 0.37]. Higher proportion of microbiologically negative children were BCG vaccinated (95% vs. 79%; p = 0.002). On multivariate analysis, microbiological positivity showed significant association with low body mass index for age (p = 0.033) and higher monocyte: lymphocyte ratio (p = 0.037). CONCLUSIONS: Low body mass index for age and higher monocyte: lymphocyte ratios were associated with microbiological confirmation in children with intrathoracic tuberculosis.

Pre-transplant cytomegalovirus immunoglobulin G antibody levels could prevent severe cytomegalovirus infections post-transplant in liver transplant recipients: Experience from a tertiary care liver centre.

BACKGROUND: Humoral immune responses in cytomegalovirus (CMV) are not studied well. Pre-transplant CMV immunoglobulin G (IgG) antibody levels (Pre-Tx IgG) could influence the occurrence of post-transplant CMV infections. OBJECTIVE: Correlation between pre-Tx IgG and post-Tx risk of acquiring CMV infection was investigated. MATERIALS AND METHODS: A total of 146 liver Tx recipients, not on CMV prophylaxis, were included. Pre-Tx IgG in donor (D) and recipient (R) were estimated and all the recipients were followed up for 1 year for CMV infections. RESULTS: D+ R+ serostatus was seen in 142 (97.3%) and D- R+ in 4 (2.7%). A total of 113 (77.4%) recipients had pre-Tx IgG of ≥250 AU/ml. Overall, post-Tx CMV infections were seen in 54 (36.9%) recipients. In 32 (59.2%) patients, CMV infection was seen during the 1st month after TX. Incidences of post-Tx CMV infection in recipients with pre-Tx IgG <250 AU/mL and ≥250 AU/mL were 42.4% and 34.5%, respectively (P = 0.99). Median viral load was significantly higher in patients with pre-Tx IgG <250 AU/ml: 4log10 (R: 2.8-6.6 log 10) copies/ml than those with ≥250 AU/ml: 2.2 log10 (R: 1.6-3.8 log10) copies/ml, P = 0.04. There was no difference in the time of occurrence of CMV infection in both the groups. Concurrent occurrence of rejection and CMV infection was seen in significantly more patients 18/54 (32.7%) than in patients without CMV infection (12/99, 12%, P = 0.002). CONCLUSIONS: Higher pre-Tx CMV IgG levels might prevent severe CMV infections post-Tx. Recipients with low pre-Tx CMV titre might be benefitted by CMV prophylaxis or aggressive pre-emptive treatment.

3q26 amplification is an effective negative triage test for LSIL: a historical prospective study.

BACKGROUND: Women with low grade squamous intraepithelial lesions (LSIL) at cervical cancer screening are currently referred for further diagnostic work up despite 80% having no precancerous lesion. The primary purpose of this study is to measure the test characteristics of 3q26 chromosome gain (3q26 gain) as a host marker of carcinogenesis in women with LSIL. A negative triage test may allow these women to be followed by cytology alone without immediate referral to colposcopy. METHODS AND FINDINGS: A historical prospective study was designed to measure 3q26 gain from the archived liquid cytology specimens diagnosed as LSIL among women attending colposcopy between 2007 and 2009. 3q26 gain was assessed on the index liquid sample; and sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were measured at immediate triage and at 6-16 months after colposcopic biopsy. The sensitivity of 3q26 gain measured at immediate triage from automated and manually reviewed tests in 65 non-pregnant unique women was 70% (95% CI: 35, 93) with a NPV of 89% (95% CI: 78, 96). The sensitivity and NPV increased to 80% (95% CI: 28, 99) and 98% (95% CI: 87, 100), respectively, when only the automated method of detecting 3q26 gain was used. CONCLUSIONS: 3q26 gain demonstrates high sensitivity and NPV as a negative triage test for women with LSIL, allowing possible guideline changes to routine surveillance instead of immediate colposcopy. Prospective studies are ongoing to establish the sensitivity, specificity, PPV and NPV of 3q26 gain for LSIL over time.

Phylogenetic analysis of 5'-UTR and P1 protein of Indian common strain of potato virus Y reveals its possible introduction in India.

The 5' untranslated region (UTR) and P1 region of the Indian strain of potato virus Y ordinary strain (PVYO) was cloned and sequenced for the first time. Database searches and multiple sequence alignment showed the highest sequence similarity with the PVYO strains of European origin. Based on the phylogenetic analysis and multiple sequence alignment, the possible evolution of PVYN from PVYO is predicted. PVYO strains from China and India were perhaps introduced into these countries from a similar geographical location. All major PVY strains available in the database can be classified into two major subgroups of North American and European origin. The Chinese and Indian PVYO strains fall within the European union subgroup suggesting a long association since potato was introduced from Europe into these countries by two separate independent events. The possible function of P1 protein in plant virus replication is suggested due to in-silico prediction of nuclear localization signal (NLS) and other phosphorylation regulatory domains at the vicinity of the NLS.

Cloning and sequencing of coat protein gene of an Indian potato leaf roll virus (PLRV) isolate and its similarity with other members of Luteoviridae.

An Indian strain of potato leaf roll virus (PLRV) was purified to generate complementary DNA corresponding to the coat protein (CP) gene. Virus cDNA was synthesized from purified viral RNA using oligo (dT)-anchor primer and virus specific primers. The viral sequence encoding the coat protein was specifically amplified by polymerase chain reaction (PCR), using specific primers bordering the CP gene. The unique amplified product thus obtained was A-T cloned into the pGEM-T Easy vector and the authenticity of the cloned gene verified by dot blot hybridization and sequence analysis. Run-way-transcripts of the cloned CP gene could detect PLRV in tissue imprints and tissue dilution. The nucleotide sequences and the deduced amino acid sequences were compared with the other PLRV isolates and found to be 97-99% identical at both the nucleotide and amino acid sequence level of other isolates. Multiple sequence alignment of deduced amino acid sequences revealed considerable homology to other luteoviruses. A nuclear localization signal located close to the N-terminus of the CP gene was predicted. This is the first report of PLRV coat protein sequence from an Indian strain.