Non-invasive prenatal determination of fetal RhD genotyping from maternal plasma: a preliminary study in Pakistan

Objectives: To determine the accuracy of the non-invasive pre-natal real-time polymerase chain reaction based fetal RhD genotyping from maternal plasma. Study Design: Cross-sectional study. Place And Duration Of Study: Juma Health Sciences Research Laboratory, The Aga Khan University Hospital, Karachi, from July to December 2008. Methodology: Cell-free plasma DNA from 21 D-negative women with D-positive spouse between 20-39 weeks of gestation was tested for the presence of exon 5 region of RhD gene using real-time polymerase chain reaction. b-globin was employed as the house-keeping gene. Sensitivity and specificity of the real-time PCR-based non-invasive fetal RhD genotyping was obtained by calculating proportion of the D-positive fetuses that were D-positive at birth as well. Results: Of the 21 D-negative women 13 and 8 neonates were determined to be D-positive and D-negative, respectively, by serologic studies on cord blood samples at birth. RhD status was correctly determined in 17 of 21 cases. There were three false-positive and one false-negative results. The sensitivity and specificity of the assay was 92.3% (95% CI: 62.1, 99.6) and 62.5% (95% CI: 25.9, 89.8), respectively. The positive and negative predictive value of the assay was 80% (95% CI: 51.4, 94.7) and 83.3% (36.5, 99.1), respectively. Conclusion: These preliminary results demonstrate the feasibility of non-invasive pre-natal diagnosis of fetal RhD status of D-negative mothers in Pakistan

The Spread of HIV in Pakistan: Bridging of the Epidemic between Populations

In the last two decades, ‘concentrated epidemics’ of human immunodeficiency virus (HIV) have established in several high risk groups in Pakistan, including Injecting Drug Users (IDUs) and among men who have sex with men (MSM). To explore the transmission patterns of HIV infection in these major high-risk groups of Pakistan, 76 HIV samples were analyzed from MSM, their female spouses and children, along with 26 samples from a previously studied cohort of IDUs. Phylogenetic analysis of HIV gag gene sequences obtained from these samples indicated a substantial degree of intermixing between the IDU and MSM populations, suggesting a bridging of HIV infection from IDUs, via MSM, to the MSM spouses and children. HIV epidemic in Pakistan is now spreading to the female spouses and offspring of bisexual MSM. HIV control and awareness programs must be refocused to include IDUs, MSM, as well as bisexual MSM, and their spouses and children

ML Phylogenetic tree of HIV-1 A Pakistani strains.

<p>ML trees where obtained with the ML method using the best fitting nucleotide substitution model. Branch lengths are scaled in nucleotide substitutions per site according to the bar at the bottom of each tree. Each tree includes the newly sequenced strains from Pakistan as well as reference strains from the HIV databases. Branches in each tree are colored according to the color legend in the figure. Pakistani sequences were labeled according to risk behavior as follows: M = male having sex with male (MSM), MS = spouses with infected partner, MC = MSM children with infected parent(s) and M-IDU = MSM who were injecting drug users. Reference strains were labeled using the HIV databases ID, which include a two letter code indicating the country of origin (<a href="http://www.hiv.lanl.gov/" target="_blank">http://www.hiv.lanl.gov/</a>).</p

Phylogenetic analysis of HIV transmission between IDUs, MSM, and MSM families.

<p>The studied population sequences were compared to the IDU sequences obtained from a previous study, which are highlighted in black. The 47 strains from MSM and M-IDU (highlighted) are shown in blue, 15 MSM spouses (MS-) are shown in green and 14 MSM children (MC-) are shown in red. Members of each family are assigned by similar digit label following the letter prefix. The numbers along the monophyletic branches correspond to bootstrap values. Branch lengths in nucleotide substitutions per site were scaled according to the bar at the bottom of the tree.</p

Demographic details of HIV gag PCR positive participants.

<p>The frequencies of all values are given in parentheses.</p