High frequency of simultaneous presence of ESBL and carbapenemase producers among nosocomial coliform isolates in Faisalabad, Pakistan.

OBJECTIVES: The objective of the current study was to find prevalence of relevant ESBL and carbapenemase producing genes in nosocomial E. coli and K. pneumoniae isolates and to check phenotypic susceptibility of all ESBL positive isolates to carbapenems. METHODS: Forty ESBL producing clinical isolates of Escherichia coli (n=33) and Klebsiella pneumoniae (n=7) were examined for the presence of ß-lactamase genes (CTX-M, CTX-M-1, 2, 3, 4 and TEM). Carbapenem resistance was checked phenotypically and by presence of blaNDM-1 gene. RESULTS: Nine (27%) were positive for CTX-M genes, and 10 (30%) for TEM among E. coli isolates. Importantly, six isolates showed co-existence of CTX-M and TEM genes. In K. pneumoniae, two (28%) isolates were positive for CTX-M and one (14%) for TEM genes. Eight (24%) E. coli and one (14%) K. pneumoniae isolates were positive for CTX-M-1. Respective figures for CTX-M-4 were three (10%) and one (14%). CTX-M-2 and CTX-M-3 groups were not represented. Twenty (50%) isolates were resistant to both imipenem and meropenem out of which only four isolates expressed blaNDM-1 gene. CONCLUSIONS: The significant presence of both ESBL and carbapenemase producers and co-existence of ESBL and carbapenemases in the same isolates is worrisome.

Rapid screening of pyogenic Staphylococcus aureus for confirmation of genus and species, methicillin resistance and virulence factors by using two novel multiplex PCR.

OBJECTIVES: Emergence of methicillin resistant Staphylococcus aureus (MRSA) is a major medical problem of current era. These bacteria are resistant to most drugs and rapid diagnosis can provide a clear guideline to clinicians. They possess specific virulence factors and relevant information can be very useful. We designed this study to develop multiplex PCRs to provide rapid information. METHODS: We studied 60 Staphylococcus aureus isolates and detected methicillin resistance by cefoxitin sensitivity and targeting of mecA gene. After initial studies with uniplex PCRs we optimized two multiplex PCRs with highly reproducible results. The first multiplex PCR was developed to confirm genus, species and methicillin resistance simultaneously, and the second multiplex PCR was for screening of virulence factors. RESULTS: We found 38.33% isolates as methicillin resistant. α -toxin, the major cytotoxic factor, was detected in 40% whereas ß-hemolysin was found in 25% cases. Panton Valentine leucocidin was detected in 8.33% and toxic shock syndrome toxin in5% cases. The results of uniplex and multiplex PCRs were highly compatible. CONCLUSIONS: These two multiplex PCRs when run simultaneously can provide vital information about methicillin resistance and virulence status of the isolate within a few hours as compared to several days needed by routine procedures.

ß-Thalassemia in Pakistan: a pilot program on prenatal diagnosis in Multan.

Prenatal diagnosis (PND) of ß-thalassemia has been underutilized in Pakistan because of a number of social and economic factors. National Institute for Biotechnology and Genetic Engineering Faisalabad in collaboration with Multan Institute of Nuclear Medicine and Radiotherapy Multan introduced free PND service for carrier couples of Multan district. Multan has a population of about 4 million. More than 170 couples registered for retrospective PND and in 2 years 105 PND were carried out through first trimester chorionic villus sampling. Almost 90% of these couples were unable to afford the cost of PND and would not have undergone the test as free service was not available. Monoplex and Multiplex Amplification Refractory Mutation System-polymerase chain reaction and genomic DNA sequencing were used for detection of IVS (intervening sequence)-I-5 (G-C), FSC (frameshift codon)-8/9 (+G), FSC-41/42 (-TTCT), IVS-I-1 (G-T), 619 bp deletion, and CD-15 (G-A) ß-globin mutations. Eighty-one percent (85/105) couples analyzed were in a consanguineous marriage. Twenty-three fetuses were found homozygous mutant and all couples opted for discontinuation of affected pregnancies. More families are registering for PND after establishment of this free and accessible PND service.

Autosomal recessive pure hair and nail ectodermal dysplasia linked to chromosome 12p11.1-q14.3 without KRTHB5 gene mutation.

Hair-nail ectodermal dysplasia (HNED; OMIM 602032) constitutes a rare subgroup of ectodermal dysplasias characterised by onychodystrophy, hypotrichosis and brittle hair. We identified a large consanguineous Pakistani family with four siblings affected by a congenital autosomal recessive form of the disease. Based on previous genetic findings in HNED we performed linkage analysis in the family using chromosome 12 markers. A genetic linkage analysis revealed a lod score of 2.92 ( = 0.0) at locus D12S368, indicating the disease gene to be located on chromosome 12. Candidate genes on chromosome 12, including the KRTHB5 gene and four additional keratin II genes, were sequenced in affected family members. Sequence analysis of the coding regions of keratin KRTHB5 gene, previously associated with a distinct clinical form of hair-nail dysplasia, revealed normal coding regions. Our study confirms linkage of a variant clinical form of hair-nail ectodermal dysplasia to chromosome 12 without any mutation in the coding sequences of the KRTHB5 gene. The results suggest this family to have either a non-coding mutation in the KRTHB5 gene, or a mutation in a yet unknown gene within the linked region on chromosome 12.

Venetoclax in the Treatment of Chronic Lymphocytic Leukemia: Evidence, Expectations, and Future Prospects.

Chronic lymphocytic leukemia (CLL) is the most common form of leukemia in the western adult population; it is also prevalent worldwide. The B cell lymphoma-2 (BCL-2) family proteins play a key role in regulating intrinsic apoptosis and, in many cancers, are the main culprits behind tumor survival and therapy resistance. Hence, the role of BCL-2 inhibitors is very beneficial in the treatment of CLL. Venetoclax is the first selective, orally bioavailable BCL-2 inhibitor. This review article discusses factors such as the pharmacokinetics, pharmacodynamics, acquired resistance to venetoclax, responders vs. non-responders in venetoclax monotherapy, and the synergistic role of venetoclax with other drugs in detail. Venetoclax is the first BH3 mimetic drug and selective BCL-2 inhibitor that has received FDA approval. This drug has proved to provide good therapeutic responses in CLL patients irrespective of the presence of adverse clinical or genetic features, including in patients with relapsed or refractory forms of CLL. We anticipate that novel combination therapies, including venetoclax and immunotherapy, will further alter the treatment landscape for patients with relapsed CLL, particularly for those with deletion 17p (del 17p) CLL, which carries a very poor prognosis.

Detection of high levels of resistance to linezolid and vancomycin in Staphylococcus aureus.

Both methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) are rapidly overcoming the current array of drugs. One hundred and fifty isolates from a hospital were studied for resistance towards linezolid and vancomycin. Fifty-four (36.0 %) isolates were MRSA. Both MRSA and MSSA showed high resistance towards linezolid when using the disc diffusion method, with the figures being 48.1 and 29.2 %, respectively. The figures for the E-test were 46.3 and 27.0 %, respectively. The vancomycin resistance was remarkable in MRSA (14.8 %), but relatively low in MSSA (3.1 %). The E-test results were 13.0 and 4.16 %, respectively. The cfr gene was detected in 78 % of linezolid-resistant isolates and the vanA operon was detected in 74 % of vancomycin-resistant isolates. This level of resistance against linezolid and vancomycin is unprecedented. These results are alarming and highlight the threat of non-treatable S. aureus strains.

Autosomal recessive transmission of a rare KRT74 variant causes hair and nail ectodermal dysplasia: allelism with dominant woolly hair/hypotrichosis.

Pure hair and nail ectodermal dysplasia (PHNED) comprises a heterogeneous group of rare heritable disorders characterized by brittle hair, hypotrichosis, onychodystrophy and micronychia. Autosomal recessive (AR) PHNED has previously been associated with mutations in either KRT85 or HOXC13 on chromosome 12p11.1-q14.3. We investigated a consanguineous Pakistani family with AR PHNED linked to the keratin gene cluster on 12p11.1 but without detectable mutations in KRT85 and HOXC13. Whole exome sequencing of affected individuals revealed homozygosity for a rare c.821T>C variant (p.Phe274Ser) in the KRT74 gene that segregates AR PHNED in the family. The transition alters the highly conserved Phe274 residue in the coil 1B domain required for long-range dimerization of keratins, suggesting that the mutation compromises the stability of intermediate filaments. Immunohistochemical (IHC) analyses confirmed a strong keratin-74 expression in the nail matrix, the nail bed and the hyponychium of mouse distal digits, as well as in normal human hair follicles. Furthermore, hair follicles and epidermis of an affected family member stained negative for Keratin-74 suggesting a loss of function mechanism mediated by the Phe274Ser substitution. Our observations show for the first time that homozygosity for a KRT74 missense variant may be associated with AR PHNED. Heterozygous KRT74 mutations have previously been associated with autosomal dominant woolly hair/hypotrichosis simplex (ADWH). Thus, our findings expand the phenotypic spectrum associated with KRT74 mutations and imply that a subtype of AR PHNED is allelic with ADWH.

A novel mutation in the Lipase H gene underlies autosomal recessive hypotrichosis and woolly hair.

Mutations in the lipase member H (LIPH) gene cause autosomal recessive hypotrichosis with woolly hair. We report herein on five consanguineous families from Pakistan segregating hypotrichosis and woolly hair. Genetic investigation using polymorphic microsatellite markers revealed homozygosity for a region spanning the HYPT7 locus on chromosome 3 in affected individuals of all five families. Sequence analysis of the LIPH gene revealed a novel nonsense mutation (p.Arg260X) associated with hypotrichosis without woolly hair in one family. In the remaining four families we identified previously described mutations in a homozygous state in affected members. These findings extend the spectrum of known LIPH mutations in the Pakistani population.

A novel mutation in lysophosphatidic acid receptor 6 gene in autosomal recessive hypotrichosis and evidence for a founder effect.

Mutations in the lysophosphatidic acid receptor 6 (LPAR6) gene cause localized autosomal recessive hypotrichosis. We report six consanguineous families from Pakistan with segregating hypotrichosis localized to the scalp. Genetic investigation using polymorphic microsatellite markers revealed homozygosity spanning the LAH3 locus on chromosome 13 in affected individuals of all six families. Sequence analysis of the LPAR6 gene showed a novel insertion resulting in a frameshift and a premature termination (p.I194FfsX11) in affected members of one family. In the remaining five families we identified a previously described missense mutation (p.G146R) in a homozygous state in affected members. The closest flanking polymorphic marker showed an identical allele size in the five families segregating with the p.G146R mutation, supporting a single origin of this variation. These findings extend the spectrum of known LPAR6 mutations and suggest a founder effect of the p.G146R mutation in the Pakistani population.

Preaxial polydactyly/triphalangeal thumb is associated with changed transcription factor-binding affinity in a family with a novel point mutation in the long-range cis-regulatory element ZRS.

A cis-regulatory sequence also known as zone of polarizing activity (ZPA) regulatory sequence (ZRS) located in intron 5 of LMBR1 is essential for expression of sonic hedgehog (SHH) in the developing posterior limb bud mesenchyme. Even though many point mutations causing preaxial duplication defects have been reported in ZRS, the underlying regulatory mechanism is still unknown. In this study, we analyzed the effect on transcription factor binding of a novel ZRS point mutation (463T>G) in a Pakistani family with preaxial polydactyly and triphalangeal thumb. Electrophoretical mobility shift assay demonstrated a marked difference between wild-type and the mutant probe, which uniquely bound one or several transcription factors extracted from Caco-2 cells. This finding supports a model in which ectopic anterior SHH expression in the developing limb results from abnormal binding of one or more transcription factors to the mutant sequence.