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Objective: Patients on chemotherapy (CT) experience a multitude of symptoms that affect nutritional intake. Therefore, we aimed to determine the symptoms that reduce food intake among patients and their perceptions towards needs of nutrition support (NS). Patients and Methods: A convenience sample of patients on CT participated in this questionnaire-based study. Results: 336 out of 445 patients on adjuvant CT participated in this survey. Among the symptoms experienced during CT, fatigue (54%) and anorexia (36%) appeared to be main contributors to decreased food intake and it was significantly more prevalent in patients with cachexia (p

This study presents a comprehensive investigation into the evolutionary trajectories of Rhipicephalus ticks (Ixodidae) through the interpretation of molecular phylogenetics, elucidating their chromatographic spectrum. The use of advanced chromatographic tools in this study explored the dynamics chemical profiling, providing valuable insights into the evolutionary history and ecological adaptations. Prevalence of Rhipicephalus ticks was 4.5% in sheep and 3.9% in goats. The ITS2 sequence of the Rhipicephalus sanguineus (OK642408) and Rhipicephalus microplus (OK642409) form a distinct clade with sequences from other countries. The 16S rRNA sequences of R. sanguineus (OK560870) clustered with sequences form three lineages, tropical, temperate, and south-eastern. The Cox I gene-identified Rhipicephalus turanicus (OK623472) and R. microplus (OK623463) form separate clades with sequences. The HPLC chromatogram of tick samples reveals a diverse array of identified hydrocarbons, explained the complex chemical composition of their exoskeletons. This analytical approach provides valuable insights into the specific hydrocarbon profiles, allowing for potential applications in species differentiation, ecological studies, and a deeper understanding of the functional roles played by hydrocarbon compounds in tick physiology. The findings revealed the potential of applying molecular phylogenetics tools with chromatography not only to enhance our understanding of tick evolution but also to inform strategies for disease control and management in regions where Rhipicephalus ticks (Ixodidae) are endemic. RESEARCH HIGHLIGHTS: Chemical mapping utilizing advanced chromatographic techniques. Scanning microscopic insights high-resolution scanning tool to observe structural and morphological features of ticks at a molecular level. Molecular phylogeny data elucidate the evolutionary relationships among tick species., (© 2024 Wiley Periodicals LLC.)

Dicrocoelium lancet flukes cause significant production loss in ruminant livestock. Although co-infection with multiple Dicrocoelium species within a host is common, techniques for studying the composition of these complex parasite communities are lacking. The pathogenicity, epidemiology, and therapeutic susceptibility of different helminth species vary, and little is known about the interactions that take place between co-infecting species and their hosts. Here, we describe the first applicationof metabarcoding deep amplicon sequencing method to studythe Dicrocoelium species in sheep and goats. First, rDNA ITS-2 sequences of four Dicrocoelium species (Dicrocoelium dendriticum, Dicrocoelium hospes, Dicrocoelium orientalis, and Dicrocoelium chinensis) were extracted from the NCBI public database. Phylogenetic analysis revealed separate clades of Dicrocoelium species; hence, molecular differentiation between each species is possible in co-infections. Second, 202 flukes belonging to seventeen host populations (morphologically verified as belonging to the Dicrocoelium genus) were evaluated to determine the deep amplicon sequencing read threshold of an individual fluke for each of the four species. The accuracy of the method in proportional quantification of samples collected from single hosts was further assessed. Overall, 198 (98.01%) flukes were confirmed as D. dendriticum and 1.98% produced no reads. The comparison of genetic distances between rDNA ITS-2 revealed 86% to 98% identity between the Dicrocoelium species. Phylogenetic analysis demonstrated a distinct clustering of species, apart from D. orientalis and D. chinensis, which sit very close to each other in a single large clade whereas D. hospes and D. dendriticum are separated into their own clade. In conclusion each sample was identified as D. dendriticum based on the proportion of MiSeq reads and validated the presence of this group of parasites in the Gilgit Baltistan and Khyber Pakhtunkhwa provinces of Pakistan. The metabarcoding deep amplicon sequencing technology and bioinformatics pathway have several potential applications, including species interactions during co-infections, identifying the host and geographical distribution of Dicrocoelium in livestock, drug therapy response evaluation and understanding of the emergence and spread of drug resistance., Competing Interests: he authors have declared that no competing interests exist., (Copyright: © 2024 Khan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Glycogen storage disease type I (GSD I) is a rare autosomal recessive inborn error of carbohydrate metabolism caused by the defects of glucose-6-phosphatase complex (G6PC). Disease causing variants in the G6PC gene, located on chromosome 17q21 result in glycogen storage disease type Ia (GSD Ia). Age of onset of GSD Ia ranges from 0.5 to 25 years with presenting features including hemorrhage, hepatic, physical and blood related abnormalities. The overall goal of proposed study was clinical and genetic characterization of GSD Ia cases from Pakistani population. This study included forty GSD Ia cases presenting with heterogeneous clinical profile including hypoglycemia, hepatomegaly, lactic acidosis i.e., pH less than 7.2, hyperuricemia, seizures, epistaxis, hypertriglyceridemia (more than180 mg/dl) and sometimes short stature. All coding exons and intron-exon boundaries of G6PC gene were screened to identify pathogenic variant in 20 patients based on availability of DNA samples and willingness to participate in molecular analysis. Pathogenic variant analysis was done using PCR-Sanger sequencing method and pathogenic effect predictions for identified variants were carried out using PROVEAN, MutationTaster, Polyphen 2, HOPE, Varsome, CADD, DANN, SIFT and HSF software. Overall, 21 variants were detected including 8 novel disease causing variants i.e., G6PC (NM_000151.4):c.71A>C (p.Gln24Pro), c.109G>C(p.Ala37Pro), c.133G>C(p.Val45Leu), c.49_50insT c.205G>A(p.Asp69Asn), c.244C>A(p.Gln82Lys) c.322A>C(p.Thr108Pro) and c.322A>C(p.Cys284Tyr) in the screened regions of G6PC gene. Out of 13 identified polymorphisms, 3 were identified in heterozygous condition while 10 were found in homozygous condition. This study revealed clinical presentation of GSD Ia cases from Pakistan and identification of novel disease-causing sequence variants in coding region and intron-exon boundaries of G6PC gene., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Gul et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Mucopolysaccharidoses (MPSs) are inherited lysosomal storage disorders (LSDs). MPSs are caused by excessive accumulation of mucopolysaccharides due to missing or deficiency of enzymes required for the degradation of specific macromolecules. MPS I-IV, MPS VI, MPS VII, and MPS IX are sub-types of mucopolysaccharidoses. Among these, MPS III (also known as Sanfilippo) and MPS IV (Morquio) syndromes are lethal and prevalent sub-types. This study aimed to identify causal genetic variants in cases of MPS III and MPS IV and characterize genotype-phenotype relations in Pakistan. We performed clinical, biochemical and genetic analysis using Whole Genome Sequencing (WGS) in 14 Pakistani families affected with MPS III or MPS IV. Patients were classified into MPS III by history of aggressive behaviors, dementia, clear cornea and into MPS IV by short trunk, short stature, reversed ratio of upper segment to lower segment with a short upper segment. Data analysis and variant selections were made based on segregation analysis, examination of known MPS III and MPS IV genes, gene function, gene expression, the pathogenicity of variants based on ACMG guidelines and in silico analysis. In total, 58 individuals from 14 families were included in the present study. Six families were clinically diagnosed with MPS III and eight families with MPS IV. WGS revealed variants in MPS-associated genes including NAGLU, SGSH, GALNS, GNPTG as well as the genes VWA3B , BTD , and GNPTG which have not previously associated with MPS. One family had causal variants in both GALNS and BTD . Accurate and early diagnosis of MPS in children represents a helpful step for designing therapeutic strategies to protect different organs from permanent damage. In addition, pre-natal screening and identification of genetic etiology will facilitate genetic counselling of the affected families. Identification of novel causal MPS genes might help identifying new targeted therapies to treat LSDs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Gul, Firasat, Schubert, Ullah, Peña, Thuesen, Gjesing, Hussain, Tufail, Saqib, Afshan and Hansen.)

More than 23 Trichuroidea species have been identified in ruminants in different parts of the world. Most are pathogenic, causing trichurosis. Trichuris adults of most species within this family have a predilection for the ceca, where they may cause damage to the epithelial wall. In the present study, Trichuris spp. from large intestine of goats were analysed based on morphological and molecular characteristics. Fifty adult worms (male = 25 and female = 25) were selected for morphometric and molecular analysis. Male Trichuris were distinguished by their longer spicules, typical spicule sheaths, and small spicules that were always completely covered by the spicule sheath. The presence of an uneverted vulva in the vagina distinguished female worms. We have performed the molecular characterisation of adult warms to identify as Trichuris skrjabini. Genetic comparison of T. skrjabini rDNA ITS2 sequences with those from other Trichuris spp. was performed to assess within and between species variation and validate the use of ITS-2 rDNA as a robust species-specific marker for T. skrjabini identification. This work provides the first report of this parasite species from Pakistan and validated species-specific marker of T. skrjabini that reduces the production potential of goats in the country., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Afshan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In some parts of the world, Dicrocoelium spp. lancet flukes cause significant production loss in pastoral livestock, and accurate diagnosis of infection is important. The aims of the present study were to describe the histopathology and to investigate the transmission patterns of Dicrocoelium amongst ten sheep and goat farms in Khyber Pakhtunkhwa and Gilgit Baltistan, Pakistan. The liver histology and indirect enzyme-linked immunosorbent assay (ELISA) analyses followed standard procedures. The liver histopathology showed intensive tissue destruction and biliary hyperplasia associated with presence of adult flukes, severe inflammatory cell infiltration, congestion of blood vessels, damaged hepatocytes, and sinusoids in the infected areas. The time of onset of infection was investigated by ELISA detection of antibodies in sheep (n = 164) and goats (n = 152). Colostral transfer of Dicrocoelium antibodies from seropositive mothers was detected in sheep and goats up to 16 weeks of age. In both sheep and goats, the estimated time of infection differed between farms and years. Infection was seen in both sheep flocks and goat herds, with high variation between flocks and herds, and the highest infection rate in lambs. Dicrocoelium infection was most prevalent in sheep and goats in September (n = 84) and August (n = 63) respectively. This study concluded Dicrocoelium causes severe inflammation and necrosis of liver tissues in sheep and goats. Colostral transfer of antibodies can be detected up to about ten weeks of age. Higher infection rates are observed during August and September in sheep than in goats, putatively due to effects of different grazing and browsing behaviors on the ingestion of ants. The results will aid in the development of effective disease control strategies to ensure optimal growth and productivity of sheep and goats., Competing Interests: Declaration of Competing Interest The authors declare no personal or financial conflicts of interest related to this work., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Purpose: Dicrocoeliosis can be an important cause of production loss in ruminants due to the cost of liver condemnation at slaughter. The aim of the present study was to determine the prevalence of Dicrocoelium infection and to predict the ecological niches and climatic variables that support dicrocoeliosis in the Himalayan ranges of Pakistan., Methods and Results: Dicrocoelium was detected in 33 of 381 liver samples and 238 of 6060 blood samples taken from sheep and goat herds in the area. The prevalence of dicrocoeliosis was higher in sheep than in goats and highest in females aged more than 3 years. An environmental risk map was created to predict active zones of transmission and showed the highest probability values in central parts of the Chitral district in the northwest of Pakistan. Climatic variables of the mean monthly diurnal temperature range (Bio2), annual precipitation (Bio12), and normalised difference vegetation index (NDVI) were found to be significantly (p < 0.05) associated with the presence of Dicrocoelium infection., Conclusion: Together, the findings of this study demonstrate the most suitable ecological niches and climatic variables influencing the risk of dicrocoeliosis in the Himalayan ranges of Pakistan. The methods and results could be used as a reference to inform the control of dicrocoeliosis in the region., (© 2022. The Author(s).)

Background: CDK5 regulatory subunit associated protein 1 like 1 (CDKAL1) encodes a tRNA modifying enzyme involved in the proper protein translation and regulation of insulin production encoded by the CDKL gene. Sequence variations in the CDKAL1 gene lead to the misreading of the Lys codon in proinsulin, resulting in decreased glucose-stimulated proinsulin production. Various polymorphic sequence variants of the CDKAL1 gene such as rs7754840, rs7756992, rs9465871, and rs10946398 are reported to be associated with type 2 diabetes mellitus and gestational diabetes mellitus (GDM) incidence. One of these single nucleotide polymorphisms i.e., rs10946398 has been reported to impact the risk of GDM and its outcomes in pregnant women of different ethnicities i.e., Egypt, Chinese, Korean, Indian, Arab, and Malaysian. Numerous findings have shown that rs10946398 overturns the regulation of CDKAL1 expression, resulting in decreased insulin production and elevated risk of GDM. However, there is no data regarding rs10946398 genotype association with GDM incidence in our population., Methodology: In this study, 47 GDM patients and 40 age-matched controls were genotyped for rs10946398 CDKAL1 variant using Tetra primer Amplification Refractory Mutation System Polymerase Chain Reaction (Tetra ARMS-PCR)., Results: Analysis of the results showed the significant association of the C allele of CDKAL1 SNP rs10946398 (χ 2  = 0.02 p = 0.001) with the risk of GDM development. Conclusively, the results support the role of SNP i.e., rs10946398 of CDKAL1 gene in GDM development in Pakistani female patients. However, future large-scale studies are needed to functionally authenticate the role of variant genotypes in the disease pathogenesis and progression., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Breast cancer patients undergoing chemotherapy (CT) experience fatigue and other side-effects. Studies exploring interventions with a plant-based, high-protein diet on fatigue and body composition are lacking. The effects of these interventions on fatigue, body mass index (BMI), and body composition were evaluated., Method: Newly diagnosed breast cancer patients who were scheduled for adjuvant CT ( n  = 103) were randomly assigned to the intervention or control group. Study outcomes included fatigue using fatigue symptom inventory and body composition using bioelectric impedance analyzer done at the start of CT, 3rd CT, and 3 weeks after CT. Linear mixed models were used to compare groups over time., Results: Fatigue decreased from 57% to 28% in the intervention group and increased from 65% to 78% in the control group ( p  < 0.001). BMI decreased by 0.7 ± 0.8 kg/m2 in the intervention group, while the decrease was 0.4 ± 1.3 kg/m2 in the control group ( p  = 0.015). Fat mass decreased in the intervention group ( p  < 0.001) and muscle mass improved in the intervention group and decreased in the control group ( p  < 0.05)., Conclusions: A plant-based, high-protein diet during CT resulted in positive changes in fatigue, BMI and body composition.

Control of tropical theileriosis, caused by the apicomplexan Theileria annulata, depends on the use of a single drug, buparvaquone, the efficacy of which is compromised by the emergence of resistance. The present study was undertaken to improve understanding of the role of mutations conferring buparvaquone resistance in T. annulata, and the effects of selection pressures on their emergence and spread. First, we investigated genetic characteristics of the cytochrome b locus associated with buparvaquone resistance in 10 susceptible and 7 resistant T. annulata isolates. The 129G (GGC) mutation was found in the Q 01 binding pocket and 253S (TCT) and 262S (TCA) mutations were identified within the Q 02 binding pocket. Next, we examined field isolates and identified cytochrome b mutations 129G (GGC), 253S (TCT) and 262S (TCA) in 21/75 buffalo-derived and 19/119 cattle-derived T. annulata isolates, providing evidence of positive selection pressure. Both hard and soft selective sweeps were identified, with striking differences between isolates. For example, 19 buffalo-derived and 7 cattle-derived isolates contained 129G (GGC) and 253S (TCT) resistance haplotypes at a high frequency, implying the emergence of resistance by a single mutation. Two buffalo-derived and 12 cattle-derived isolates contained equally high frequencies of 129G (GGC), 253S (TCT), 129G (GGC)/253S (TCT) and 262S (TCA) resistance haplotypes, implying the emergence of resistance by pre-existing or recurrent mutations. Phylogenetic analysis further revealed that 9 and 21 unique haplotypes in buffalo and cattle-derived isolates were present in a single lineage, suggesting a single origin. We propose that animal migration between farms is an important factor in the spread of buparvaquone resistance in endemic regions of Pakistan. The overall outcomes will be useful in understanding how drug resistance emerges and spreads, and this information will help design strategies to optimise the use and lifespan of the single most drug use to control tropical theileriosis., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2022. Published by Elsevier Ltd.)

Inherited retinal dystrophies (IRDs) are a heterogeneous group of degenerative disorders of the retina. Retinitis Pigmentosa (RP) is a common type of IRD that causes night blindness and loss of peripheral vision and may progress to blindness. Mutations in more than 300 genes have been associated with syndromic and non-syndromic IRDs. Recessive forms are more frequent in populations where endogamy is a social preference, such as Pakistan. The aim of this study was to identify molecular determinants of IRDs with the common presentation of night blindness in consanguineous Pakistani families. This study included nine consanguineous IRD-affected families that presented autosomal recessive inheritance of the night blindness phenotype. DNA was extracted from blood samples. Targeted exome sequencing of 344 known genes for retinal dystrophies was performed. Screening of nine affected families revealed two novel (c.5571&#95;5576delinsCTAGATand c.471dup in EYS and SPATA7 genes, respectively) and six reported pathogenic mutations (c.304C>A, c.187C>T, c.1560C>A, c.547C>T, c.109del and c.9911&#95;11550del in PDE6A, USH2A, USH2A, NMNAT1, PAX6 and ALMS1 genes, respectively) segregating with disease phenotype in each respective family. Molecular determinants of hereditary retinal dystrophies were identified in all screened families. Identification of novel variants aid future diagnosis of retinal dystrophies and help to provide genetic counseling to affected families.

Background: Primary congenital glaucoma (PCG) is a heterogeneous rare recessively inherited disorder prevalent in regions with high consanguinity. Disease phenotype is associated with increased intra ocular pressure and is a major cause of childhood blindness. Sequence variations in Cytochrome P450 1B1 (CYP1B1) gene are a major cause of PCG. Current study was conducted to screen CYP1B1 gene in highly consanguineous PCG affected families from Pakistani population consistent with the autosomal recessive pattern of PCG inheritance., Methods: For this study, patients and controls (clinically unaffected individuals of each family) from 25 consanguineous families belonging to Punjab, Baluchistan and Khyber Pakhtunkhwa, Pakistan were recruited through ophthalmologists. DNA was isolated from collected blood samples. Genetic screening of CYP1B1 gene was done for all enrolled families. In-silico analysis was performed to identify and predict the potential disease-causing variations., Results: Pathogenicity screening revealed sequence variants segregating with disease phenotype in homozygous or compound heterozygous form in eleven out of 25 analyzed families. We identified a total of sixteen disease causing variants among which five frameshift i.e., c.629dup (p.Gly211Argfs*13), c.287dup (p.Leu97Alafs*127), c.662dup (p.Arg222Profs*2), c.758&#95;759insA (p.Val254Glyfs*73) and c.789dup (p.Leu264Alafs*63), two silent c.1314G>A, c.771T>G and six missense variations c.457C>G (p.Arg153Gly), c.516C>A (p.Ser172Arg), c.722T>A (p.Val241Glu), c.740T>A (p.Leu247Gln), c.1263T>A (p.Phe421Leu), and c.724G>C (p.Asp242His) are previously un reported. However two frameshift c.868dup (p.Arg290Profs*37), c.247del (p.Asp83Thrfs*12) and one missense variant c.732G>A (p.Met244Ile), is previously reported. Furthermore, six polymorphisms c.1347T>C, c.2244&#95;2245insT, c.355G>T, c.1294G>C, c.1358A>G and c.142C>G were also identified. In the intronic region, a novel silent polymorphism i.e., g.35710&#95;35711insT was found in homozygous state. All the newly detected disease-causing variants were negative in 96 ethnically matched controls., Conclusion: Among twenty-five screened families, eight families (PCG50, 52-54, 58, 59, 63 and 67) were segregating disease causing variants in recessive manner. Two families (PCG049 and PCG062) had compound heterozygosity. Our data confirms genetic heterogeneity of PCG in Pakistani population however we did not find molecular variants segregating with PCG in fifteen families in coding exons and intron-exon boundaries of CYP1B1 gene. Genetic counseling was provided to families to refrain from practicing consanguinity and perform premarital screening as a PCG control measure in upcoming generations., Competing Interests: The authors have declared that no competing interests exist.

Wilson's disease (WD) is an autosomal recessive disorder, resulting from variations in ATP7B gene. Clinical heterogeneity, including neuropsychiatric and hepatic manifestations over a large range of age groups make diagnosis difficult. Most of WD patients suffer severe disabilities and even die. So, overall goal of proposed study is the genetic and clinical characterization of Wilson's disease cases from Pakistani population. Clinical data was collected, and patients were investigated for variations in selected ATP7B exons using PCR based Sanger sequencing. Pathogenic effect predictions for detected variants were carried out using PROVEAN, MutationTaster2, and HSF software's. Clinical heterogeneity was observed in patients including reduced serum ceruloplasmin, signs of chronic liver damage and raised 24 h urinary copper excretion. Mean age of onset was 11.3 years. Kayser-Fleischer rings were present in 75% of cases. About 82.5% patients belonged to inbred families. Patients having neurological disorder were above 12 years of age. Total ten variants in analyzed region of ATP7B gene, including a reported variation (p. L227Yfs*35) were found in patients. The study also identified 4 putative novel synonymous variants (c.251A>C, c.15T>A, c.6T>C, c.238C>T) and 5 reported polymorphisms (c.83C>A, c.39&#95;40insCGGCG, p.V456L, c.39&#95;40insCGCCG and c.1544-53A>C). Reliable understanding of clinical presentations and genotype-phenotype correlation provide insight to function and structure of ATP7B and may assist in disease prognosis and family counseling. The study revealed clinical presentation of Pakistani WD cases and identification of sequence variants in screened region of ATP7B., Competing Interests: The authors have no conflict of interest to declare.

Background: Interleukin-4 (IL-4) plays a central role in the humoral immune defense against nematode parasite infections, inducing IgE switch and regulation of worm expulsion from the intestines. The present study aimed to investigate the polymorphisms in IL-4 gene and their association with socio-demographic and environmental factors among patients with gastrointestinal complaints., Method: The screened population comprised 305 patients aged 3-50 years from Rawalpindi and Jhelum districts of Pakistan. A well-prepared questionnaire was administered to collect data on socio-demographic and environmental factors. The data were analyzed by using multiple logistic regression models. Molecular analysis was done on 88 confirmed cases passing worms and eggs in stool by using PCR to amplify IL-4 gene., Results: The result showed higher GI nematodes prevalence in Rawalpindi 34.87% and Jhelum 23.1% among gastrointestinal patients. The multivariate logistic regression model showed significantly (p<0.05) increased risk of infection in participants who were residing in rural areas (OR=321.94; 22.5), having poor economic status (OR=0.34), consuming raw/unwashed vegetables (OR=1.73; 15.39) and did not practice handwashing (OR=2.77; OR=0.30). Sequence analysis showed three novel polymorphisms at SNP g.704&#95;705 ins T, g.3763&#95;3764 ins AC and g.3792 G >A in patients with acute severe infections. Two known polymorphisms SNPs g.8455A>G and g.8492C>A were found in the intron region., Conclusion: IL-4 gene polymorphisms showed disease susceptibility and consuming raw/unwashed vegetables, poor handwashing practices and poor economic status were the most associated factors with the disease., (© 2022 Afshan K et al.)

Malaria is the world's fatal parasitic disease. The ability to quickly and accurately identify malaria infection in challenging environments is crucial to allow efficient administration of the best treatment regime for human patients. If those techniques are accessible and efficient, global detection of Plasmodium species will become more sensitive, allowing faster and more precise action to be taken for disease control strategies. Recent advances in technology have enhanced our ability to diagnose different species of Plasmodium parasites with greater sensitivity and specificity. This literature review provides a summary and discussion of the current methods for the diagnosis and identification of Plasmodium spp. in human blood samples. So far not a single method is precise, but advanced technologies give consistent identification of a Plasmodium infection in endemic regions. By using the power of the recent methods, we can provide a broader understanding of the multiplicity of infection and or transmission dynamics of Plasmodium spp. This will result in improved disease control strategies, better-informed policy, and effective treatment for malaria-positive patients., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

Background: Autosomal recessive corneal hereditary endothelial dystrophy (CHED) is a rare congenital disorder of cornea. Mutations in SLC4A11 gene are associated with CHED phenotype. CHED is also an early feature of Harboyan syndrome. The aim of the present study was to identify genetic mutations in the SLC4A11 gene in CHED cases belonging to inbred Pakistani families. Furthermore, all homozygous mutation carriers were investigated for hearing deficit., Methods and Results: This study included consanguineous CHED families presented at Al-Shifa Trust Eye Hospital, Rawalpindi, Pakistan from June 2018 to September 2018. DNA was extracted from blood samples. Direct sequencing of SLC4A11 gene was performed. All identified variants were evaluated by in silico programs i.e., SIFT, PolyPhen-2, and MutationTaster. Pathogenicity of the two identified splice site variants was analyzed by Human Splicing Finder and MaxEnt Scan. Screening of five CHED families revealed a total of three previously un reported (p.Arg128Gly, c.2241-2A > T and c.1898-2A > C in family CHED19, CHED22 and CHED26 respectively) and two already reported homozygous disease causing variants (p.Arg869Cys and p.Val824Met in family CHED24 and CHED25 respectively) as predicted by mutation taster. All of these variants segregated with disease phenotype and were not detected in controls., Conclusion: Affected individuals of the five CHED families screened in this study had the disease due to SLC4A11 mutations and progressing to Harboyan syndrome. Identification of previously unreported mutations aid to heterogeneity of SLC4A11 and CHED pathogenesis as well as helped to provide genetic counseling to affected families., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Objective: To explore epidemiology, clinical profiles and contribution of reproductive and non-reproductive risk factors in breast cancer development., Methods: The case-control study was conducted from October 2017 to March 2018 at Quaid-i-Azam University, Islamabad, Pakistan, and comprised breast cancer patients and age-matched controls recruited from the Bahawalpur Institute of Nuclear Medicine and Oncology, and the Bahawal Victoria Hospital, Bahawalpur. Socio-demographic data, family history of cancer, reproductive health and lifestyle factors were recorded using a structured questionnaire. Data was analysed using SPSS 21 and Stata/IC 14.1., Results: Of the 326 women, 163(50%) each were cases and controls. The mean age for both the groups was identical at 46.04±10.62 years. Positive family history and hypertension were significantly linked to increased breast cancer risk (p<0.05), while intense physical activity, increased anthropometric measurements and breastfeeding per child in months were inversely associated with the risk (p<0.05)., Conclusions: Established risk factors for breast cancer were reaffirmed.

Objective: Intrahepatic Cholestasis of Pregnancy (ICP) is a rare pregnancy specific disorder. Genetic variants of ABCB4 gene increase ICP risk. This study was conducted to determine frequency of ICP cases presented at a tertiary care hospital in Rawalpindi, Pakistan and to screen for genetic variants of exon 6 and 14 of ABCB4 gene in ICP cases., Methods: This analytical study included ICP patients presenting at Department of Gynaecology and Obstetrics, Holy Family Hospital Rawalpindi, from February 2017 to May 2017. Sanger's sequencing was performed using genomic DNA extracted from blood samples of patients and controls., Results: Twenty pregnant women out of 1150 (1.74%) had ICP and were enrolled during study period. Overall (19/20) 95% patients had pruritus and among them (8/20) 40%, (4/20) 20% and (2/20) 10% had a history of miscarriages, stillbirths and familial ICP respectively. Genetic analysis revealed an already reported variant i.e., c.504C>T in exon 6 in thirteen patients and a novel variant i.e., c.1686A>G in exon 14 in five patients. Both variants were not present in controls. In silico analysis suggested that both variants might affect pre-mRNA splicing of ABCB4 transcript., Conclusions: ICP had a frequency of 1.74% among pregnant women. Identification of a novel heterozygous variant in five patients and an already reported variant in thirteen patients reaffirms genetic heterogeneity and role of ABCB4 in ICP etiology.

Lancet liver flukes of the genus Dicrocoelium (Trematoda: Digenea) are recognised parasites of domestic and wild herbivores. The aim of the present study was to confirm the species identity of Dicrocoeliid flukes collected from the Chitral valley in the Himalayan ranges of Pakistan. The morphology of 48 flukes belonging to eight host populations was examined; but overlapping traits prevented accurate species designation. Phylogenetic comparison of published D. dendriticum ribosomal cistron DNA, and cytochrome oxidase-1 (COX-1) mitochondrial DNA sequences with those from D. chinensis was performed to assess within and between species variation and re-affirm the use of species-specific single nucleotide polymorphism markers. PCR and sequencing of 34 corresponding fragments of ribosomal DNA and 14 corresponding fragments of mitochondrial DNA from the Chitral valley flukes, revealed 10 and 4 unique haplotypes, respectively. These confirmed for the first time the molecular species identity of Pakistani lancet liver flukes as D. dendriticum. This work provides a preliminary illustration of a phylogenetic approach that could be developed to study the ecology, biological diversity, and epidemiology of Dicrocoeliid lancet flukes when they are identified in new settings., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Latent transforming growth factor beta binding protein 2 (LTBP2) plays a critical role in the development of connective tissue structure and function. Mutations in gene encoding LTBP2 are known to cause syndromic and a non-syndromic microspherophakia. Here, we present a 'first' report of genetic linkage of microspherophakia (MSP) to LTBP2 locus in a large consanguineous Pakistani family with four affected individuals in three loops. Using polymorphic microsatellite markers, haplotypes and linkage analysis, the diseased phenotype in MSP001 family was mapped to the LTBP2 gene. A maximum two point Logarithm of the odds (LOD) score of 4.16 was obtained with marker D14S284 at θ =0. Mutational analysis of exon 36 of LTBP2 using Sanger's sequencing did not reveal any previously reported mutations. Further analysis of the remaining exons are required to identify the causative variant.

Introduction: Little is known about the genetic and morphological characters of Paramphistomum epiclitum. For the first time in Pakistan, adult flukes were morphologically characterized and the sequence variation in the nuclear ribosomal DNA (rDNA) region, including the first internal transcribed spacers (ITS1) and the 5.8S gene of the Paramphistomum epiclitum were studied., Methods: Adult amphistomes were examined by light microscopy and sequences of ITS1 and 5.88S rDNA genes were obtained., Results: Twenty adult flukes were measured, 13.17 ± 1.19 mm in length and 5.28 ± 1.34 mm in width. Seventeen adult flukes were sequenced and high sequence variability was observed in 5' end of ITS1 region. The 5.8S and 3' end of ITS1 sequences had 100% identity among the samples. A comparative analysis revealed that different types and numbers of repeats were found within each ITS1 region. The 3' end of ITS1 region from P. epiclitum showed 98% homology with P. cervi from China and formed a subclade with genetic distance of 0.1663. The 5.8S gene showed 100% identity within Paramphistomidae family and formed a sub-clade with P. epiclitum, P. leydeni, P. cervi and Cotylophoron cotylophorum species isolated from China, India and Uruguay., Conclusion: This work provides new information on morphological identity and genetics of P. epiclitum from Pakistan.

Aim: The aim of the study was to define social determinants, causes and trends in child mortality from 1990 to 2013 in Pakistan. Understanding social determinants, causes and temporal trends in child mortality can inform strategies aimed at improving child health in low and middle income countries., Methods: We characterised temporal trends and social determinants of child mortality in Pakistan using national demographic health survey data (1990-1991, 2006-2007 and 2012-2013). We analysed national data to generate regional estimates of health programme effectiveness., Results: The annual rates of reduction for child mortality between 1990 and 2013 were estimated as follows: neonatal (-0.33% per annum), post-neonatal (3.13% per annum), infant (0.9% per annum), child (2.47% per annum) and under 5 (1.19% per annum). Bivariate analyses of 2013 data showed that living in Punjab or Balochistan province, belonging to lowest wealth quintile, lack of maternal education, previous birth interval < 2 years, first birth order and below average birth size were associated (p < 0.05) with greater risk of child mortality in Pakistan. Common factors associated with child mortality were fever, diarrhoea and pneumonia, while among stillbirths, intrapartum asphyxia, unexplained antepartum and antepartum maternal disorders were most frequent., Conclusion: Child survival strategies should integrate leading biosocial indicators and causes of death. Further research is needed to define the role(s) of social factors in child health and survival. These data should inform the implementation of cost-effective interventions for child survival and advance targeting of interventions to populations at increased risk of child mortality., (© 2019 Paediatrics and Child Health Division (The Royal Australasian College of Physicians).)

Background: Fascioliasis has never been considered a public health concern in Pakistan, although the increasing numbers of human cases reported in south Asia need a re-consideration in the country. The current study aimed to find the seroprevalence of human fascioliasis, associated risk factors and its relationship with liver enzymes as biomarkers of pathogenicity., Methods: The cross-sectional study was conducted in different districts of Punjab region from May 2014 to August 2016. A total of 546 respondents were screened by using enzyme-linked immunosorbent assay (ELISA) and serum biochemical tests., Results: Higher seroprevalence was recorded in Muzaffargarh (6.2%) and Bhara kahu (5.9%), while low infection rate in Gujranwala (1.1%) and Islamabad (1.5%). The results of multiple logistic regression analysis showed rural inhabitants (OR=7.9, 95%CI: 2.5-24.8), females (OR=3.5, 95%CI: 1.7-7.1), family size 3-7 (OR=1.7, 95%CI: 1.0-2.9) and socioeconomic condition (OR=3.9, 95%CI: 1.5-10.4) were the significantly (p<0.005) associated risk factors with disease. The results of liver enzymes i.e. aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transferase and cholesterol levels were significantly (p=0.001) elevated and associated with fascioliasis pathogenicity., Conclusion: The higher prevalence recorded may explain with Fasciola IgG antibodies for both active and past infections and cross reactivity of the assay with other helminthes., (© 2020 Afshan K et al.)

Fanconi anemia (FA) is a recessive disorder that predispose to bone marrow failure and multiple congenital anomalies in affected individuals worldwide. To date, 22 FA genes are known to harbor sequence variations in disease phenotype. Among these, mutations in the FANCA gene are associated with 60% to 70% of FA cases. The aim of the present study was to screen FA cases belonging to consanguineous Pakistani families for selected exons of FANCA gene which are known mutational hotspots for Asian populations. Blood samples were collected from 20 FA cases and 20 controls. RNA was extracted and cDNA was synthesized from blood samples of cases. DNA was extracted from blood samples of cases and ethnically matched healthy controls. Sanger's sequencing of the nine selected exons of FANCA gene in FA cases revealed 19 genetic alterations of which 15 were single nucleotide variants, three were insertions and one was microdeletion. Of the total 19 sequence changes, 13 were novel and six were previously reported. All identified variants were evaluated by computational programs including SIFT, PolyPhen-2 and Mutation taster. Seven out of 20 analyzed patients were carrying homozygous novel sequence variations, predicted to be associated with FA. These disease associated novel variants were not detected in ethnically matched controls and depict genetic heterogeneity of disease., (© 2019 Japanese Teratology Society.)

Objectives: To investigate the seroprevalence and associated risk factors of entamoeba histolytica among patients with gastrointestinal complaints, and to measure the eventual changes in serum biochemical parameters to reflect its pathogenicity., Methods: The cross-sectional study was conducted in different hospitals of Potohar region in Punjab province and in the Khyber Pakhtunkhwa province of Pakistan from September 2015 to February 2017, and comprised individuals of either gender belonging to diverse backgrounds, inhabiting different areas of the country. The patients were enrolled from among those who visited outpatient departments with complaints of vague abdominal pain, nausea, vomiting, indigestion and diarrhoea. Blood samples were screened by using enzyme-linked immunosorbent assay and serum biochemical tests. Data was analysed using SPSS 20., Results: Of the 356 subjects, 238(66.9%) were females and 118 (33.1%) were males. The overall mean age was 33.4}11.05 years. Seroprevalence of entamoeba histolytica was 356(73%). The infection rate did not differ significantly (p>0.05) among cities, while the highest infection was recorded in Islamabad 91(25.5%). The participants in rural areas had 2.16-fold higher risk of infection compared to urban areas, while the lowest risk of infection among people aged 50years compared to those aged 40-49 years (p=0.04). The amoebiasis was significantly associated with eating unwashed raw vegetables and average toilet facilities. Among clinical complications, haemodynamic changes, jaundice, vomiting, haemoglobin level, loose motion, intolerance to oral feeding, and history of antibiotics were significant associated variables (p<0.05 each). Significant elevation in alkaline phosphatase, aspartate aminotransferase, total protein and globulin levels were positively associated with amoebiasis (p<0.01 each)., Conclusions: In entamoeba histolytica -positive patients ,serum biochemical level was found elevated and the risk factors determined were eating unwashed vegetables, toilet facilities, age, locality, jaundice, vomiting, haemoglobin level, loose motion, intolerance to oral feeding, and history of antibiotics.

Primary congenital glaucoma (PCG) causes blindness in early age. It has an autosomal recessive pattern of inheritance, hence is more prevalent in populations with frequent consanguineous marriages that occur in the Pakistani population. Mutations in the CYP1B1 gene are commonly associated with PCG. The aim of the present study was to identify genetic mutations in the CYP1B1 gene in PCG cases belonging to 38 Pakistani families. DNA was extracted using blood samples collected from all enrolled patients, their available unaffected family members and controls. Direct sequencing of the CYP1B1 gene revealed a novel 3' splice acceptor site causative variant segregating in an autosomal recessive manner in a large consanguineous family with four PCG-affected individuals. The novel variant was not detected in 93 ethnically matched controls. Furthermore, four already reported mutations, including p.G61E, p.R355X, p.R368H, and p.R390H were also detected in patients belonging to nine different families. All identified causative variants were evaluated by computational programs, that is, SIFT, PolyPhen-2, and MutationTaster. Pathogenicity of the novel splice site variant identified in this study was analyzed by Human Splicing Finder and MaxEntScan. Ten out of 38 families with PCG had the disease due to CYP1B1 mutations, suggesting CYP1B1 was contributing to PCG in these Pakistani patients. Identification of this novel 3' splice acceptor site variant in intron 2 is the first report for the CYP1B1 gene contributing to genetic heterogeneity of disease., (© 2018 Japanese Teratology Society.)

Background: Globally 3 million stillbirths occur per year, and Pakistan is ranked 3 rd among the countries having the highest burden. Despite being a major public health problem, efforts to reduce this figure are insufficient., Objective: The aim of the study was to identify and measure the inequalities in stillbirth associated risk factors, causes and fertility risk behaviors., Methods: Data were derived from the Pakistan Demographic and Health Surveys (PDHS) 1990-2013. Inequalities on determinants were evaluated using rate differences and rate ratios; time trends computed with annualized reduction rate (ARR)., Results: Overall ARR determined for stillbirth was -12.52 percent per annum. The high ARR were recorded for mothers age <20, urban areas, educated mothers and for highest wealth quintile. The relative inequalities were most pronounced for wealth quintiles, education and age of mothers. Stillbirth causes were unexplained antepartum (33%), unexplained intrapartum (21%), intrapartum asphyxia (21%) and antepartum maternal disorders (19%). The high fertility risk behavior was found in mothers with age >34 and birth order >3., Conclusion: The study concluded that to achieve gain in child survival, there is need to promote antenatal care, birth spacing, and family planning programs in developing countries.

Soil transmitted helminths are causing significant morbidity worldwide and the most common infection is Ascaris lumbricoides in developing countries. The present study aimed to determine the immuno-epidemiological status of ascariasis among patients with gastrointestinal complaints and to identify the associated risk factors and eventual changes in serum biochemical parameters to reflect its pathogenicity. This study was conducted on 700 respondents aged between 5-45 years. A total of 356 patients participated in an enzymelinked immunosorbent assay (ELISA) study to determine anti-Ascaris IgG levels and biochemical parameters. The overall seroprevalence was 58.4%, with 100% sensitivity and 84.4% specificity of the assay. The infection was highest among the 21-28 year age group (14.0%), and ascariasis was found to be not significantly (P>0.05) different between the age groups. The results showed that the risk of ascariasis was significantly (P<0.05) increased in individuals who had no contact with soil (OR=4.6, 95% CI: 1.9-10.8), eating unwashed vegetables one month prior to the study (OR=2.7, 95% CI: 1.4-5.2), eating mixed food (OR=2.4, 95% CI: 1.2-4.7), drinking pressure pump water (OR=3.4, 95% CI: 1.9-6.1), and those who had no complain of vomiting (OR=3.1, 95% CI: 1.6-5.8) and nausea (OR=1.9, 95% CI: 1.1-3.2). The results showed significantly (P<0.05) elevated level of serum alanine aminotransferase, alkaline phosphatase, serum cholesterol, total protein and globulin in anti-Ascaris IgG positive cases than the control group. The study concluded that patients who visited health care centres with gastrointestinal complain were at higher risk of ascariasis as compared to other diseases. In conclusion epidemiological studies are needed to establish baseline data for public health authorities in order to plan and implement health education programs to reduce the impact of the disease.

Intestinal parasitic infections (IPIs) are a major cause of morbidity worldwide and have been described as an important public health concern. The present study aimed to determine the prevalence and identification of risk factors associated with IPIs among 3-15 years old school age children residing in Mandi Bahauddin, Pakistan from 2011- 2013. A cross sectional school-based study was conducted using a structured pre-tested questionnaire. Anthropometric tools and stool tests were used to obtain epidemiological and disease data. The direct wet mount preperation in saline/iodine/haematoxylin stain and Kato-Katz methods were used for stool examination. Data were analysed using appropriate descriptive, univariate and multivariable logistic regression methods. Of the 1,434 children studied (mean age of 8.6±3.6 years) the overall prevalence rate for intestinal parasitic infections was found to be 33.3%. Children infected with single parasite accounted for 27.6% and 5.7% were detected with poly-parasitism. The study showed that helminths (21.4%) were more prevalent than protozoans (17.9%). Ascaris lumbricoides (17.5%), Giardia lamblia (9.8%), Entamoeba histolytica (8.2%), Hymenolepis nana (2.0%), Trichuris trichiura (1.3%) and Taenia saginata (0.7%) were identified in children living in irrigated areas. The multiple logistic regression model indicated that age of the child, gender, family size, source of drinking water, type of milk used, house condition, feeding habit, personal hygiene and socioeconomic status were significantly (p<0.05) associated with the IPIs. Intestinal parasites were prevalent in varying magnitude among the schoolchildren located in irrigated areas. We conclude that there is a need for mass scale campaigns to create awareness regarding health and hygiene in children, and the need for development of effective poverty control programmes because deworming alone is not adequate to control parasitic infections.

An understanding of the ABO and Rh blood group systems is important for blood transfusions and is also pertinent due to their potential association with certain morbidities and susceptibilities to infections. To investigate the diversity and differentiation of the ABO and Rh loci in Middle Eastern populations, data from twelve representative Middle Eastern populations were analyzed. Six populations were in conformity with Hardy-Weinberg equilibrium at the ABO locus. The pooled heterozygosity at both loci was calculated to be highest in the sample from Jordan and lowest in Bahrain. Heterogeneity was pronounced in the Northern compared to the Southern Middle Eastern populations. Overall, the absolute gene diversity was 0.0046 and gene differentiation was calculated to be 0.0100. Genetic diversity of the studied loci across all populations (HT) was estimated to be 0.4594, while the diversity within the populations (HS) was 0.4548. Nei's genetic distance analyses revealed highest affinities between the populations of Kuwait and Qatar, Oman and Yemen, and between Qatar and the United Arab Emirates. These results were displayed through a UGPMA dendrogram and principal component analyses, which established clustering of certain populations. Clinal trends of the allelic systems were observed by generating contour maps that allow a detailed appreciation of the distributions of alleles across the geography of the Arabian Peninsula and the Middle East. Taken together, these analyses are helpful in understanding the differentiation of blood group loci and for designing prospective studies for establishing the associations of these loci with health variables in the populations studied.

Artemisia plant genus, natural inhabitant of northern Punjab Pakistan, is well known for its anthelmintic properties; many Artemisia species have not been so far scientifically proved. The aim of this study was to assess in vitro anthelmintic activity of Artemisia indica and Artemisia roxburghiana against mixed infection of gastrointestinal nematodes in small ruminants. This study is first scientifically proven study on anthelmintic activity of A. indica and A. roxburghiana. Five different concentrations (50, 25, 12.5, 6.25 and 3.75 mg/mL) accompanied by negative control (PBS) and positive control (albendazole, 10%) were used to carry out the egg hatch inhibition assay, larval mortality assay and adult worm mortality assay. The Baermann technique was used first time in larval mortality assay and proved to be effective. The results revealed that methanolic extracts of both A. indica and A. roxburghiana, showed maximum anthelmintic activity at concentration of 50 mg/ml by egg hatch inhibition (85±21.2; 80±28.3), larvae mortality (18±2.8; 17±4.2) and adult worm mortality (8.5±2.1; 8±2.8) assays. However, at concentration of 50 mg/ml both plant extracts in comparison to albendazole showed statistically insignificant (p≤0.05) results. The A. indica showed higher anthelmintic activity at all concentrations as compared to A. roburghiana. It has been concluded both plants exhibit anthelmintic activity and further evaluation of these plants should be carried out to purify the active ingredients for anthelmintic activity. Moreover, the decoctions of these plants could be used to GINs after confirming anthelmintic properties through in vivo.

Fascioliasis is an important food-borne parasitic disease caused by the two trematode species, Fasciola hepatica and Fasciola gigantica. The phenotypic features of fasciolid adults and eggs infecting buffaloes inhabiting the Central Punjab area, Pakistan, have been studied to characterize fasciolid populations involved. Morphometric analyses were made with a computer image analysis system (CIAS) applied on the basis of standardized measurements. Since it is the first study of this kind undertaken in Pakistan, the results are compared to pure fasciolid populations: (a) F. hepatica from the European Mediterranean area; and (b) F. gigantica from Burkina Faso; i.e. geographical areas where both species do not co-exist. Only parasites obtained from bovines were used. The multivariate analysis showed that the characteristics, including egg morphometrics, of fasciolids from Central Punjab, Pakistan, are between F. hepatica and F. gigantica standard populations. Similarly, the morphometric measurements of fasciolid eggs from Central Punjab are also between F. hepatica and F. gigantica standard populations. These results demonstrate the existence of fasciolid intermediate forms in endemic areas in Pakistan.

Large areas of the province of Punjab, Pakistan are endemic for fascioliasis, resulting in high economic losses due to livestock infection but also affecting humans directly. The prevalence in livestock varies pronouncedly in space and time (1-70%). Climatic factors influencing fascioliasis presence and potential spread were analysed based on data from five meteorological stations during 1990-2010. Variables such as wet days (Mt), water-budget-based system (Wb-bs) indices and the normalized difference vegetation index (NDVI), were obtained and correlated with geographical distribution, seasonality patterns and the two-decade evolution of fascioliasis in livestock throughout the province. The combined approach by these three indices proved to furnish a useful tool to analyse the complex epidemiology that includes (i) sheep-goats and cattlebuffaloes presenting different immunological responses to fasciolids; (ii) overlap of Fasciola hepatica and F. gigantica; (iii) co-existence of highlands and lowlands in the area studied; and (iv) disease transmission following bi-seasonality with one peak related to natural rainfall and another peak related to man-made irrigation. Results suggest a human infection situation of concern and illustrate how climate and anthropogenic environment modifications influence both geographical distribution and seasonality of fascioliasis risks. Increased fascioliasis risk throughout the Punjab plain and its decrease in the northern highlands of the province became evident during the study period. The high risk in the lowlands is worrying given that Punjab province largely consists of low-altitude, highly irrigated plains. The importance of livestock in this province makes it essential to prioritise adequate control measures. An annual treatment scheme to control the disease is recommended to be applied throughout the whole province.

We describe efforts towards introducing infection control (IC) practices and establishment of antimicrobial resistance (AMR) surveillance in a public sector hospital in Pakistan. The study was conducted in an eight-bed intensive care unit. IC principles, introduced through interactive sessions, were used as an intervention and their impact was observed by conducting surveillance for ventilator-associated pneumonia (VAP) before and after the intervention. Respiratory isolates of VAP patients in the period after intervention were screened for AMR, and empiric antibiotic at the time of admission was compared with the antimicrobial sensitivity pattern reported. VAP rates were high in general and declined in the period after intervention, although the difference was not significant. Of 37 VAP patients in the period after intervention, 68% had more than one clinically significant organism isolated from the respiratory specimen. Acinetobacter spp. were isolated from 76% of patients and Pseudomonas aeruginosa from 43%. All Acinetobacter spp. and 72% P. aeruginosa were multidrug resistant. The mean stay of the nosocomially infected patients was significantly higher than for the uninfected group (6.5 vs. 2.1 days, P<0.001). Our study suggests IC education needs to be supplemented by a hospital system that facilitates IC practices and development of surveillance programmes.