HIV tropism switch in archived DNA of HIV-HCV subjects successfully treated with direct-acting antivirals for HCV infection

We described short-term HIV tropism changes occurring in peripheral blood mononuclear cells and the correlations with HIV DNA value in HIV-HCV co-infected patients cured for HCV disease and with undetectable HIV viremia or residual viremia (RV). Plasma HIV RNA, cellular HIV DNA and tropism were evaluated pre-HCV treatment (baseline, BL) and at 12(T1) and 24(T2) weeks after HCV treatment start. V3 sequences were interpreted using Geno2pheno and classified as R5 only if all three sequences had an FPR ≥ 10% and as X4 when at least one replicate sequence had an FPR < 10%. Forty-nine patients (21 with X4 and 28 with R5 virus) were enrolled. Five X4 patients and 9 R5 subjects experienced at least one tropism change,11 with RV:1/5 patients with X4 infection at BL switched at T1 versus 8/9 in the R5 group (p = 0.022977) and the difference was confirmed in subjects with RV (p = 0.02);6/9 R5 patients switching at T1 confirmed the tropism change at T2. No significant differences in HIV DNA values between patients with RV starting with a R5 or X4 tropism and experienced tropism switch or not were found. Short-term tropism switch involved almost a third of patients, in all but three cases with HIV RV. Being R5 at BL is associated to a higher instability, expressed as number of tropism changes and confirmed switch at T2.

CYTOMEGALOVIRUS DNA CONCENTRATION IN BIOLOGICAL SAMPLES AS A KEY TO THE DIAGNOSIS OF CMV PNEUMONIA IN HIV-INFECTED PATIENTS

According to examination and follow-up results of 5485 HIV-positive hospitalized patients (3333 of which were diagnosed with AIDS) we have identified the frequency of clinically evident CMV-infection as well as the frequency and character of CMV related lung disease. Statistically significant correlation between viral load, degree of immunosuppression, CMV replication rate and CMV pneumonia development risk has been determined. Qualitative PCR assay for CMV DNA in plasma and respiratory samples was found to have high sensitivity and low specificity for diagnosing CMV-pneumonia. We identified quantitative PCR CMV DNA values in blood cells, plasma, bronchoalveolar lavage, bronchi samples and sputum that confirm the diagnosis of CMV pneumonia with 95% and 99% probability, and exclude CMV related lung damage in HIV patients with 90% and 99% probability.

Comparative study of HBV DNA levels in HBeAg positive and negative female patients suffering from chronic hepatitis b in child bearing age.

To compare HBV DNA values in HBeAg positive and negative female patients suffering from chronic hepatitis B of child bearing age. Study Design: Cross sectional study. Setting: Hospitals attached to Peshawar Medical College, Peshawar after taking ethical approval from the institution’s ethical board. Period: October 2014 to March 2015. Materials and Methods: Blood samples were collected from 100 chronic hepatitis B females of child bearing age. Quantitative HBeAg was done by ELISA and HBV DNA values were estimated by PCR. The data obtained was statistically analyzed by using the Statistical Package for the Social Sciences (SPSS) version 17 and statistical significance between the variables was calculated using Fisher’s exact test. Results: The mean age of the sample was 28.69±6.83 years. A positive correlation (p=0.000) was found in HBeAg positive status and younger age group females (17-30). 84 cases were HBeAg negative but 42/84 had high levels of circulating Hepatitis B virus (2000 IU/ml). Conclusion: The present study established a positive association between HBeAg positive status and high HBV DNA values. However a statistically positive association was observed between HBeAg negative patients and high HBV DNA values (HBV DNA ≥2000IU/ml). Objective: To compare HBV DNA values in HBeAg positive and negative female patients suffering from chronic hepatitis B of child bearing age. Methodology: This cross sectional study was conducted from October 2014 to March 2015 in Hospitals attached to Peshawar Medical College, Peshawar after taking ethical approval from the institution’s ethical board. Blood samples were collected from 100 chronic hepatitis B females of child bearing age. Quantitative HBeAg was done by ELISA and HBV DNA values were estimated by PCR. The data obtained was statistically analyzed by using the Statistical Package for the Social Sciences (SPSS) version 17 and statistical significance between the variables was calculated using Fisher’s exact test. Results: The mean age of the sample was 28.69±6.83 years. A positive correlation (p=0.000) was found in HBeAg positive status and younger age group females (17-30). 84 cases were HBeAg negative but 42/84 had high levels of circulating Hepatitis B virus (2000 IU/ml). Conclusion: The present study established a positive association between HBeAg positive status and high HBV DNA values. However a statistically positive association was observed between HBeAg negative patients and high HBV DNA values (HBV DNA ≥2000IU/ml). Key Words: Hepatitis B virus, HBeAg, HBV DNA

DNA IMAGE CYTOMETRY IN PROGNOSTICATION OF COLORECTAL CANCER: PRACTICAL CONSIDERATIONS OF THE TECHNIQUE AND INTERPRETATION OF THE HISTOGRAMS

The role of DNA content as a prognostic factor in colorectal cancer (CRC) is highly controversial. Some of these controversies are due to purely technical reasons, e.g. variable practices in interpreting the DNA histograms, which is problematic particularly in advanced cases. In this report, we give a detailed account on various options how these histograms could be optimally interpreted, with the idea of establishing the potential value of DNA image cytometry in prognosis and in selection of proper treatment. Material consists of nuclei isolated from 50 ƒĘm paraffin sections from 160 patients with stage II, III or IV CRC diagnosed, treated and followed-up in our clinic. The nuclei were stained with the Feulgen stain. Nuclear DNA was measured using computer-assisted image cytometry. We applied 4 different approaches to analyse the DNA histograms: 1) appearance of the histogram (ABCDE approach), 2) range of DNA values, 3) peak evaluation, and 4) events present at high DNA values. Intra-observer reproducibility of these four histogram interpretation was 89%, 95%, 96%, and 100%, respectively. We depicted selected histograms to illustrate the four analytical approaches in cases with different stages of CRC, with variable disease outcome. In our analysis, the range of DNA values was the best prognosticator, i.e., the tumours with the widest histograms had the most ominous prognosis. These data implicate that DNA cytometry based on isolated nuclei is valuable in predicting the prognosis of CRC. Different interpretation techniques differed in their reproducibility, but the method showing the best prognostic value also had high reproducibility in our analysis.

c-myc oncogene gene dosage, serum CEA and CA-15.3 antigen levels, and cellular DNA values in relation to ex vivo chemosensitivity of primary human breast cancer.

A pilot study on relationships of selected molecular factors (c-myc oncogene average gene copy numbers (AGCN); serum CEA and CA 15.3 antigen levels; tumor cells' DNA values), to the ex vivo chemosensitivity of primary female human breast cancer in a modified adenosine triphosphate cell viability chemosensitivity assay (ATP-CVA), was performed. Four drug combinations were tested. A group of 75 cases of female primary breast cancer was assessed. Numerous correlations were found among molecular factors tested but none, with the exception of tumor grading, of these reflected ex vivo chemosensitivity of tumors tested. The results suggest that the parameters tested may not be important factors related to adjuvant chemoresponsiveness of primary human breast cancer to tested drug combinations.

Nuclear DNA amount in pure species and hybrid willows (Salix): a flow cytometric investigation

Flow cytometry (FCM) has been used to estimate the nuclear DNA content of 11 Salix species and 5 hybrids. One hundred and sixty nine individuals were studied including 159 individuals from a sequence of 32 communities along a stretch of river in France and 10 individuals from French and English collections for comparison. Isolated nuclei were stained with propidium iodide. FCM was a significantly more practical and rapid technique than that of establishing the karyotype to survey many samples of Salix for variation in ploidy. The 2C DNA amounts for diploid species ranged from 0.76 to 0.98 pg, and tetraploid values ranged from 1.62 to 1.80 pg. The DNA values were consistent with the known ploidy levels. With the exception of a doubtful Salix xquercifolia, ploidy levels and DNA amounts of hybrids were intermediate compared with those of their parents. Intraspecific variation of nuclear DNA values including instrumental variation was low (i.e., 6-11% at the same ploidy level). FCM appeared to be an accurate tool for determination of Salix triploid hybrids. However, it remains limited concerning hybrids from crosses between species of the same ploidy level. Results suggest that natural hybridization might not be frequent in the communities studied, although they have been subject to disturbance. Previous overestimates of hybridization frequency in willows were probably due to misinterpretation of the effects of the environment on Salix spp. morphology; however, the extent and mechanisms of introgression in the genus remain to be further investigated. Key words: flow cytometry, Salix, hybridization, nuclear DNA content, riparian vegetation, disturbance.

A chromosome atlas and interspecific – intergenic index for Lotus and Tetragonolobus (Fabaceae)

Basic chromosome numbers in Lotus are x = 5, 6, and 7. It is considered that evolution has proceeded in the genus by means of a descending aneuploid series from an eight-chromosomed ancestor. Chromosome numbers for species of Tetragonolobus are based on x = 7. Somatic chromosome numbers are reported for 108 species and 38 varieties. The chromosome numbers for five species (L. hamatus Greene, 2n = 14, L. haydonii (Orcutt) Greene, 2n = 14, L. hintoniorum B.L. Turner, 2n = 14, L. mearnsii Britton, 2n = 14, L. utahensis Ottley, 2n = 14) and seven varieties (L. argophyllus (Gray) Green var. argenteus Dunkle, 2n = 14, L. dendroideus var. traskiae (Eastwood ex Noddin) Isely, 2n = 14, L. heermanii (Durand et Hilgard) Greene var. orbicularis (Gray) Isely, 2n = 14, L. junceus (Benth.) Greene var. biolettii (Greene) Ottley, 2n = 14, L. strigosus var. hirtellus (Greene) Ottley, 2n = 14, L. strigosus var. tomentellus (Greene) Isely, 2n = 14, L. uliginosus ssp. vestitus (Lange) A. Pedersen, 2n = 12) are reported for the first time. Natural diploid, tetraploid, and hexaploid plants are reported for L. alpinus. Several species are reported as possessing B chromosomes. Mixoploidy is reported to occur in three species (L. alpinus, L. glacialis, L. glareosus). In addition, chromosome numbers are given for plants regenerated from calluses grown in tissue culture having both heteroploidy, euploidy, and mixoploidy. Root nodules are reported with tetraploid and octoploid cells in addition to the normal number of chromosomes. Trisomie series have been partially developed in L. tenuis and L. uliginosus. Polytene chromosomes were observed in suspensor cells of three species of Lotus. Feulgen cytophotometric measurements, to determine the DNA nuclear content, were made for 16 species of Lotus and one species of Tetragonolobus. The majority of the studies in Lotus concern the economic species L. corniculatus, L. tenuis, and L. uliginosus. Interspecific hybridization was carried out in different combinations between diploids, autoploids, and amphidiploids. Intergeneric hybrids were attempted by somatic hybridization, protoplast fusion, and asymmetric hybridization between Lotus and other species (Glycine max, Medicago sativa, Oryza sativa). Key words: chromosome numbers, DNA values, Fabaceae, Lotus species, interspecific hybrids, intergeneric hybrids, Tetragonolobus.