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Objective: To assess the reliability of different red blood cell indices-based formulae in the indexes formula in differential diagnosis of beta thalassemia trait and iron deficiency anaemia., Methods: This cross-sectional study was conducted between January and October 2015 in Dera Ismail Khan in the Khyber Pakhtunkhwa province of Pakistan. Patients of beta thalassemia trait and iron deficiency anaemia were registered irrespective of age and gender. About 5 mL of blood was taken from each patient to analyse different red cell parameters like red blood cell count, haemoglobin, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, and red cell distribution width. Five formulae were used to discriminate between the two conditions. These were red cell distribution width index, Shine and Lal index, Mentzer index, Srivastava index, and the Green and King index. Sensitivity, specificity, positive and negative predictive values and Youden's index of all the indices were calculated., Results: Of the 800 patients, 230(29%) had beta thalassemia trait and 570(71%) had iron deficiency anaemia. The red cell distribution width index appeared be a reliable index in discriminating between beta thalassemia trait and iron deficiency anaemia with sensitivity and specificity of 100% and 93% respectively., Conclusions: The red cell distribution width index was the most consistent index for differentiating between beta thalassemia trait and iron deficiency anaemia. IIt could be used as a screening index for beta thalassemia trait in areas where haemoglobin electrophoresis facility is unavailable.

Hepatitis C virus (HCV) chronically infects almost 2% of world's population. Chronic infection can lead to liver failure and hepatocellular carcinoma (HCC). Approximately 10% of the Pakistani population is infected with HCV and type 3 is the most prevalent genotype with 75-90% prevalence. In this study we have developed transiently expressing cell culture based system for the expression of HCV non-structural NS3, NS3-4A and NS4A proteins of genotype 3a. HCV non-structural genes NS3, NS3-4A and NS4A were cloned in to pFLAG-CMV2 and pEGFP-C1vectors. All vectors were transfected separately to Huh-7 cells and their protein expression was analyzed by Western blot and immunofluorescence. All proteins were expressed correctly and in the transfection we have obtained 42-70% efficiency for all clones. This system can be used for the development of novel antiviral strategies to inhibit the viral replication, to study apoptosis pathways induced by HCV, for the evaluation of vaccine candidates and also to study the role of HCV different signaling pathways.

Background: Plasmodium vivax is the most prevalent malaria species in Pakistan, with a distribution that coincides with Plasmodium falciparum in many parts of the country. Both species are likely exposed to drug pressure from a number of anti-malarials including chloroquine, sulphadoxine-pyrimethamine (SP), and artemisinin combination therapy, yet little is known regarding the effects of drug pressure on parasite genes associated with drug resistance. The aims of this study were to determine the prevalence of polymorphisms in the SP resistance-associated genes pvdhfr, pvdhps and chloroquine resistance-associated gene pvmdr1 in P. vivax isolates collected from across the country., Methods: In 2011, 801 microscopically confirmed malaria-parasite positive filter paper blood samples were collected at 14 sites representing four provinces and the capital city of Islamabad. Species-specific polymerase chain reaction (PCR) was used to identify human Plasmodium species infection. PCR-positive P. vivax isolates were subjected to sequencing of pvdhfr, pvdhps and pvmdr1 and to real-time PCR analysis to assess pvmdr1 copy number variation., Results: Of the 801 samples, 536 were determined to be P. vivax, 128 were P. falciparum, 43 were mixed vivax/falciparum infections and 94 were PCR-negative for Plasmodium infection. Of PCR-positive P. vivax samples, 372 were selected for sequence analysis. Seventy-six of the isolates (23%) were double mutant at positions S58R and S117N in pvdhfr. Additionally, two mutations at positions N50I and S93H were observed in 55 (15%) and 24 (7%) of samples, respectively. Three 18 base pair insertion-deletions (indels) were observed in pvdhfr, with two insertions at different nucleotide positions in 36 isolates and deletions in 10. Ninety-two percent of samples contained the pvdhps (S382/A383G/K512/A553/V585) SAKAV wild type haplotype. For pvmdr1, all isolates were wild type at position Y976F and 335 (98%) carried the mutation at codon F1076L. All isolates harboured single copies of the pvmdr1 gene., Conclusions: The prevalence of mutations associated with SP resistance in P. vivax is low in Pakistan. The high prevalence of P. vivax mutant pvmdr1 codon F1076L indicates that efficacy of chloroquine plus primaquine could be in danger of being compromised, but further studies are required to assess the clinical relevance of this observation. These findings will serve as a baseline for further monitoring of drug-resistant P. vivax malaria in Pakistan.

Background: Few studies have been conducted in Pakistan to determine the efficacy of chloroquine and sulphadoxine-pyrimethamine (SP), which remain in use as treatment for Plasmodium vivax and in combination with artesunate to treat Plasmodium falciparum, respectively. In this study, samples from several sites across Pakistan were characterized to determine prevalence of molecular resistance markers in the P. falciparum chloroquine resistance transporter (pfcrt), multidrug resistance (pfmdr1), dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes and the origin of chloroquine-resistant P. falciparum parasites., Methods: Microscopy-confirmed malaria parasite-positive blood samples from 801 patients across the country were collected in 2011. Of these, 171 infections were identified by polymerase chain reaction (PCR) as P. falciparum and analysed by pyrosequencing for mutations conferring chloroquine resistance (pfcrt codons 72-76), multidrug resistance (pfmdr1 N86Y, Y184F, S1034C, N1042D and D1246Y), pyrimethamine resistance (pfdhfr, C50R, N51I, C59R, S108N and I164L) and sulphadoxine resistance (pfdhps, S436A, A437G, K540E, A581G and A613T/S). pfmdr1 gene copy number variation was determined by real-time PCR, and microsatellites flanking the pfcrt locus were typed to determine the origin of the chloroquine-resistant haplotype., Results: The pfcrt K76T mutation was found in all samples as part of the S72/V73/M74/N75/T76 (SVMNT) haplotype. Microsatellites flanking pfcrt showed high similarity to the signature found in India and Papua New Guinea. pfmdr1 N86Y was found in 20% of samples and all samples harboured a single copy of the pfmdr1 gene. The pfdhfr double mutation C59R + S108N was present in 87% of samples while the pfdhfr triple mutant (N51I + C59R + S108N) was not detected. Pfdhps A437G was found in 60% of samples. Pure pfdhps K540E was rare, at 4%, but mixed genotype 540 K/E was found in 77% of samples. Similarly, pure pfdhps A581G was found in 4% of the isolates while mixed 581A/G was found in 39% of samples., Conclusions: These results suggest an emerging problem with multidrug resistant P. falciparum in Pakistan. The chloroquine resistance genotype has reached complete fixation in the population, with a microsatellite pattern indicative of a selective sweep. Moreover, the prevalence of mutations in both pfdhfr and pfdhps, albeit without the presence of the pfdhfr triple mutant, indicates that continued monitoring is warranted to assess whether SP remains efficacious as a partner drug for artesunate for the treatment of P. falciparum.

Background: Both Plasmodium vivax and Plasmodium falciparum are prevalent in Pakistan, yet up-to-date data on the epidemiology of malaria in Pakistan are not available. This study was undertaken to determine the current prevalence and distribution of Plasmodium species across the country., Methods: A malariometric population survey was conducted in 2011 using blood samples collected from 801 febrile patients of all ages in four provinces and the capital city of Islamabad. Microscopically confirmed Plasmodium-positive blood samples were reconfirmed by polymerase chain reaction (PCR). Confirmed parasite-positive samples were subjected to species-specific PCR capable of detecting four species of human malaria., Results: Of the 707 PCR-positive samples, 128 (18%) were P. falciparum, 536 (76%) were P. vivax, and 43 (6%) were mixed P. falciparum and P. vivax. Ninety-four microscopy-positive samples were PCR-negative, and Plasmodium malariae and Plasmodium ovale were not detected. Prevalence of P. vivax ranged from 2.4% in Punjab Province to 10.8% in Sindh Province and prevalence of P. falciparum ranged from 0.1% in Islamabad to 3.8% in Balochistan., Conclusions: Plasmodium infections in Pakistan are largely attributed to P. vivax but P. falciparum and mixed species infections are also prevalent. In addition, regional variation in the prevalence and species composition of malaria is high.

Background: Intrauterine growth restriction is a major neonatal health issue. Maternal factors have been found to have greater impact on IUGR. Studying these factors can help in reducing the mortality and morbidity associated with IUGR., Methods: This case-control study was conducted at the department of Paediatrics Post-graduate medical institute Lady Reading Hospital Peshawar from March 2008-April 2009. Small-for-gestational age (SGA, i.e., IUGR cases and n = 200) live born babies were compared with appropriate-for-gestational age (AGA, i.e., controls and n = 200) babies. Information regarding socio-demographics of mothers, gestational age and birth weight of baby, maternal clinical characteristics, and medical and obstetric complications during pregnancy was recorded on a pre-designed proforma. Data analysis was done through SPSS-16. To find the maternal factors associated with the intrauterine growth restriction, multivariable logistic regression was used. We also did two different sets of logistic regression analysis for Symmetric and Asymmetric SGA babies as Cases., Results: After adjusting for other variables in the multivariable model we found that the mothers of IUGR babies were of younger age (OR = 0.8, CI = 0.7-0.9), were poor (OR = 2.5, CI = 1.4-4.4) and underweight (OR = 3.5, CI = 1.1-5.7) and had anaemia (OR = 2.7, CI = 1.3-5.4) in the index pregnancy, and had history of Previous IUGR birth (OR = 9.7, CI = 3.3-18.3) and placenta previa (OR = 3.2, CI = 1.1-6.6). There was an interaction between pregnancy induced hypertension and parity of mother with a primary-para mother with pregnancy induced hypertension (PIH) having an increased risk for IUGR babies (OR = 10.1, CI = 1.0-23.2)., Conclusion: The studied factors need special attention in hospital based settings in order to improve the perinatal outcome in IUGR babies.

Background: Very preterm babies are important group of paediatric babies who require special attention. These babies are known to have increased risk of morbidity and mortality. Studying the morbidity and mortality pattern for this important paediatric group can help in better understanding of their care in the hospital settings. Objective of the study was to compare the mortality and morbidity pattern in Small-for-gestational age and appropriate-for-gestational age very preterm babies. This hospital based prospective (cohort) study was conducted at the department of Paediatrics, Postgraduate Medical Institute, Lady Reading Hospital, Peshawar from March 2008 to April 2009., Methods: One hundred Small-for-gestational age (SGA) live born very preterm babies were compared with 100 appropriate-for-gestational age (AGA) very preterm babies having similar gestational ages. Information regarding gestational age, birth weight, mortality, and morbidity (in terms of various biochemical and clinical markers) were recorded on a pre-designed questionnaire. Data analysis was done using SPSS version 15. Results were interpreted in terms of descriptive (mean, proportions, standard deviation) and inferential statistical tests (with p-values)., Results: There was no difference between the two groups (SGA vs. AGA) with regards to gestational age and gender of the babies The mean weight of SGA babies was significantly lower as compared to AGA babies (1.1 +/- 0.16 Kg Vs 1.5 +/- 0.2 Kg; p = 0.001). As compared to AGA babies, the SGA babies had a higher mortality (40% vs. 22%, p = 0.006), and higher morbidity in terms of hyperbilirubinaemia (67% vs. 51%, p = 0.02) and hypocalcaemia (24% vs. 10%, p=0.02). The difference in the mortality between the two groups was more prominent in babies with gestational age < or =31 weeks (71.4% for SGA as compared to 39.3% for AGA ery preterm babies with gestational age < or =31 weeks)., Conclusion: Very preterm SGA infants have significantly higher mortality and morbidity in comparison to the AGA babies. In deciding for therapeutic management of these babies, they need special attention in terms of factors as gestational age and the biochemical markers, to improve the outcome for these babies in the hospital settings.