Detection of rifampicin resistance of Mycobacterium tuberculosis using multiplex allele specific polymerase chain reaction (MAS-PCR) in Pakistan.

Drug resistance in tuberculosis (TB) is a major public health challenge in developing countries such as Pakistan. Multiplex allele specific polymerase chain reaction (MAS-PCR) is a DNA amplification method that could contribute to rapid detection and control of drug resistant tuberculosis (DR-TB) in Pakistan. The purpose of this study was to test the utility of MAS-PCR to detect resistance in Pakistan. Drug susceptibility testing (DST) was used to identify rifampicin resistant and susceptible clinical isolates from TB cases in Pakistan. MAS-PCR was used to detect the most frequent mutations in the gene rpoB among 213 resistant and 37 susceptible isolates. Among 213 clinical isolates, MAS-PCR identified mutation D435Y (Asp435Tyr) in 24 (11.3%) cases, H445Y (His445Tyr) in 14 (6.6%), S450L (Ser450Leu) in 124 (58.2%) and S450W (Ser450Trp) in 18 (8.4%) cases. MAS-PCR did not detect known mutations in 33 (15.5%) cases. Among 12 cases, a novel mutation at codon 434 (Met434Ile) and a common variant at codon 435 (Asp435Tyr) was detected in rpoB gene which is indicative of double mutation. In 4 isolates, a novel mutation at codon 432 (Gln432Pro) was identified. In an additional 4 isolates, mutations Met434Val and His445Asn were identified. Moreover, a mutation in rpoB (Leu452Pro) was found in 5 isolates. DNA sequencing confirmed the absence of mutations in rpoB in the 8 remaining isolates. MAS-PCR had 88.3% sensitivity and 100% specificity using DST as the reference, which suggested that this method could be implemented as an initial marker for screening of multi-drug resistant tuberculosis (MDR-TB) in Pakistan.

Pattern of Drug Resistance and Risk Factors Associated with Development of Drug Resistant Mycobacterium tuberculosis in Pakistan.

BACKGROUND: Drug resistant tuberculosis (DR-TB) is a major public health problem in developing countries such as Pakistan. OBJECTIVE: The current study was conducted to assess the frequency of drug resistant tuberculosis including multi drug resistance (MDR- TB) as well as risk factors for development of DR-TB, in Punjab, Pakistan. METHODOLOGY: Drug susceptibility testing (DST) was performed, using proportion method, for 2367 culture positive Mycobacterium tuberculosis (MTB) cases that were enrolled from January 2012 to December 2013 in the province of Punjab, Pakistan, against first-line anti-tuberculosis drugs. The data was analyzed using statistical software; SPSS version 18. RESULTS: Out of 2367 isolates, 273 (11.5%) were resistant to at least one anti-TB drug, while 221 (9.3%) showed MDR- TB. Risk factors for development of MDR-TB were early age (ranges between 10-25 years) and previously treated TB patients. CONCLUSION: DR-TB is a considerable problem in Pakistan. Major risk factors are previous history of TB treatment and younger age group. It emphasizes the need for effective TB control Program in the country.

Prevalence of plasmid-mediated AmpC ß-lactamases in Escherichia coli and Klebsiella pneumonia at tertiary care hospital of Islamabad, Pakistan.

Enterobacteriaceae produces AmpC ß-lactamases that make them resistant to commonly used antibiotics. AmpC ß-lactamases can be chromosomal-mediated or plasmid-mediated AmpC ß-lactamases (PABLs). The present study was undertaken to determine the occurrence of PABLs production in clinical isolates in Escherichia coli and Klebsiella pneumoniae. Among 1328 culture positive samples, 511 isolates were identified as E. coli (81.02%, n = 414) and K. pneumonia (18.98%, n = 97). Cefoxitin resistance was observed in E. coli (19.57%, n = 81) and K. pneumoniae (22.68%, n = 22). Out of these cefoxitin resistant isolates, 40.74% (n = 33) E. coli and 54.55% (n = 12) K. pneumoniae came out to be PABL producers. Prevalence of both PABLs and ESBLs in E. coli and K. pneumoniae was 29.24% (n = 8) and 47% (n = 5), respectively. Isolates coproducing PABLs and ESBL exhibited increased minimum inhibitory concentrations (MICs) for selected cephalosporins. This study documented a high frequency of PABLs producing isolates from hospital which may lead to serious therapeutic problem.

Establishment of an efficient callus induction and plant regeneration system in Pakistani wheat (Triticum aestivum) cultivars

Four commercially grown wheat varieties of Pakistan, namely Inqilab-91, Chakwal-97, Tatara and Manthar were used for this investigation. For callus induction different concentrations of 2,4-Dichlorophenoxyaceticacid (2,4-D) along with 0.1 mg/L of Kinetin were evaluated. For regeneration initially different concentrations of Indole-3-Acetic Acid (IAA) and 6-BenzylAminoPurine (BAP) were tested. Best hormone combinations were further subjected to Kinetin and 6-ã-ã-dimethylallylaminopurine (2iP). For Inqilab-91, Chakwal-97 and Manthar, 3 mg/L of 2,4-D was found optimum, which induced 83.25 percent, 77.75 percent and 95.20 percent of embryogenic calli, respectively. Maximum callus induction (97.18 percent) was observed in Tatara when 2 mg/L of 2,4-D was used. As regard to regeneration, Inqilab-91, Chakwal-97 and Manthar showed maximum regeneration on media containing 0.1 mg/L IAA, 0.4 mg/L Kinetin and 0.5 mg/L 2iP, regenerating 87.25 percent, 81.75 percent and 68.75 percent respectively. For Tatara maximum regeneration of 12.25 percent was obtained on 0.1 mg/L IAA and 2 mg/L of BAP. Presently optimized regeneration method holds promise for facilitating the deployment of agronomical important trait through genetic transformation for the improvement of this important food crop.

Phytoaccumulation prospects of cadmium and zinc by mycorrhizal plant species growing in industrially polluted soils.

The natural vegetation growing along a wastewater channel was subjected to analyze the uptake of Cadmium (Cd) and Zinc (Zn) and their subsequent accumulation in aboveground and underground plant parts. Species which were mycorrhizal and growing in soils receiving industrially contaminated wastewater were collected along with their rhizospheric soil samples. The nearby uncontaminated control (reference) area was also subjected to sampling on similar pattern for comparison. Both Cd and Zn concentrations were significantly higher in soils of the study area as compared to the reference site. Five plant species i.e. Desmostachya bipinnata, Dichanthium annulatum, Malvastrum coromandelianum, Saccharum bengalense, and Trifolium alexandrinum were analyzed for metal uptake. The maximum phytoaccumulation of Cd was observed in Desmostachya bipinnata (20.41 microg g(-1)) and Dichanthium annulatum (15.22 microg g(-1)) for shoot and root tissues, respectively. However, Malvastrum coromandelianum revealed maximum Zn accumulation for both the shoot and the root tissues (134 and 140 mug g(-1), respectively). The examination of cleared and stained roots of the plants from both the areas studied revealed that all of them were colonized to a lesser or a greater degree by arbuscular mycorrhizal (AM) fungi. The Cd hyperaccumulating grasses i.e. Desmostachya bipinnata and Dichanthium annulatum, from study area had smaller root:shoot (R/S) ratio as compared to those growing on reference area indicating a negative pressure of soil metal contamination. The lower R/S ratio in the mycorrhizal roots observed was probably due to increased AM infection and its mediatory role in soil plant transfer of heavy metals. Furthermore, comparatively lower soil pH values in the study areas may have played a key role in making the overall phytoavailability of both the metals. Consequently variations in Cd and Zn tissue concentration among species were observed that also indicate the phytoaccumulation potential of the native species.

Rearing the cotton bollworm, Helicoverpa armigera, on a tapioca-based artificial diet.

The impact of a tapioca-based artificial diet on the developmental rate, life history parameters, and fertility was examined over five consecutive generations for the cotton bollworm, Helicoverpa armigera Hubner (Lepidoptera: Noctuidae), a highly polyphagous pest of many agricultural crops. The study showed that when fed the tapioca-based artificial diet during larval stage, larval and pupal developmental period, percent pupating, pupal weight, emergence rate of male and female, longevity, fecundity and hatching were non-significantly different than that of the control agar-based artificial diet. Moreover, the cost to rear on tapioca-based diet approached 2.13 times less than the cost of rearing on the agar-based artificial diet. These results demonstrate the effectiveness and potential cost savings of the tapioca-based artificial diet for rearing H. armigera.

Soil solarization: a safe, affective and practicable technique for the control of soil born fungi and nematodes.

A technique i.e., Soil Solarization and Amendments (neem, chicken farmyard manure, farmyard manure and biokhad viz synthetic bio fertilizer), towards the natural cropping system has been evaluated for its effectiveness and practicability at the National Agricultural Research Center Islamabad Pakistan. Soil solarization and amendments were analyzed as a control measure against soil born fungi and nematodes. Eight weeks of solarization resulted in about 11 degrees C increase in the soil temperature. This increase in soil temperature caused a reduction of about 70 to 80% in the fungal population and about 99% in nematode population at various depths. Neem and Biokhad amendments were proved synergistic for solarization and also improved the properties of soil in the benefit of crop plants. Fusarium sp., Macrophomina phyaseolina and Verticillium sp. of fungi and Tylenchus sp., Haplolaimus sp., Xiphenema sp. and almost all of the parasitic nematodes were significantly (p<0.01) controlled. It was found that even after 40 days the solarized plots contain significantly less number of fungi and nematodes as compared to the nonsolarized plots, which confirmed the durability of this process.

Passive immunization using purified IgYs against infectious bursal disease of chickens in Pakistan.

Infectious bursal disease (IBD) is an acute and highly contagious disease of young chickens caused by Birnavirus. Mortality of infected birds can be best prevented if injected with antibodies. The present study was an attempt to raise specific hyper-immune polyclonal antibodies against IBD virus in Pakistan. Commercial layers divided into four groups were injected with IBD vaccine subcutaneously according to four different treatment regimens. Eggs were collected daily and antibodies were purified from yolk with dextran sulphate. Titers of antibodies in serum and yolk were evaluated with enzyme linked immunosorbant assay and agar gel precipitation test. Antibody titers were significantly higher in yolk than serum. Eggs collected at 28 days post-vaccination had maximum antibody titers. Of treatment regimens, T3 was found to be most effective for hyperimmunization. Lyophilized antibodies stored at 4 degrees C did not lose their activity till the end of experiment. IBD virus infected birds were injected with purified antibodies which induced 92% recovery as compared to control birds. The study implicates that the purified antibodies may be useful as a therapeutic agent to cure IBD infected birds.