Rani Faryal

Exploitation of Aspergillus terreus for the production of natural statins

The fungus Aspergillus (A.) terreus has dominated the biological production of the “blockbuster” drugs known as statins. The statins are a class of drugs that inhibit HMG-CoA reductase and lead to lower cholesterol production. The statins were initially discovered in fungi and for many years fungi were the sole source for the statins. At present, novel chemically synthesised statins are produced as inspired by the naturally occurring statin molecules. The isolation of the natural statins, compactin, mevastatin and lovastatin from A. terreus represents one of the great achievements of industrial microbiology. Here we review the discovery of statins, along with strategies that have been applied to scale up their production by A. terreus strains. The strategies encompass many of the techniques available in industrial microbiology and include the optimization of media and fermentation conditions, the improvement of strains through classical mutagenesis, induced genetic manipulation and the use of statistical design.

Dengue Virus Serotypes Circulating in Khyber Pakhtunkhwa Province, Pakistan, 2013-2015

From 2013 to 2015, the National Institute of Health, Pakistan, received 1,270 blood samples of suspected dengue cases reported from inpatient and outpatient departments of various hospitals in Khyber Pakhtunkhwa (KPK) province. In this study, we determined the circulating dengue virus (DENV) serotypes using real-time reverse transcriptase (RT)-PCR to understand the serotype-based epidemiology of DENV. All four serotypes (DENV-1 [6%], DENV-2 [33%], DENV-3 [47%], and DENV-4 [0.1%]) were found circulating during the study period. Our findings suggest the need for an active surveillance system coupled with the laboratory diagnosis, especially in the chronic endemic areas of the country. Public awareness programs are needed for effective control and prevention of outbreaks in the future.

Preliminary Seroepidemiological survey of dengue infections in Pakistan, 2009-2014

BackgroundDengue virus is the causative agent of dengue fever, a vector borne infection which causes self-limiting to life threatening disease in humans. A sero-epidemiological study was conducted to understand the current epidemiology of dengue virus in Pakistan which is now known as a dengue endemic country after its first reported outbreak in 1994.MethodsTo investigate the prevalence of dengue virus in Pakistan during 2009-2014, a total of 9,493 blood samples were screened for the detection of anti-dengue IgM antibodies using ELISA. Clinical and demographic features available with hospital records were reviewed to ascertain mortalities related to dengue hemorrhagic shock syndrome.ResultsOut of 9,493 samples tested, 37% (3,504) were found positive for anti-dengue IgM antibodies. Of the seropositive cases, 73.6% (2,578/3,504) were male and 26.4% (926/3,504) were female. The highest number (382/929; 41.1%) of sero-positive cases was observed among the individuals of age group 31-40 years. The highest number of symptomatic cases was reported in October (46%; 4,400/9,493), and the highest number of sero-positive cases among symptomatic cases was observed in November (45.7%; 806/1,764). Mean annual patient incidence (MAPI) during 2009-2014 in Pakistan remained 0.30 with the highest annual patient incidence (11.03) found in Islamabad. According to the available medical case record, 472 dengue related deaths were reported during 2009-2014.ConclusionThe data from earlier reports in Pakistan described the dengue virus incidence from limited areas of the country. Our findings are important considering the testing of clinical samples at a larger scale covering patients of vast geographical regions and warrants timely implementation of dengue vector surveillance and control programs.Trial registration numberIt is an epidemiological research study, so trial registration is not required.

Inhibition of secreted aspartyl proteinase activity in biofilms of Candida species by mycogenic silver nanoparticles

Abstract Candida species are pleomorphic, commensal fungi associated with candidiasis. The extracellular hydrolytic-secreted aspartyl proteinases are recognized as chief agents for pathogenesis of Candida species, involved in the degradation of proteins and adhesion during biofilm formation. This study aimed at exploring inhibitory effect of mycogenic silver nanoparticles (Ag NPs) against C. albicans and non-albicans’ biofilm growth and aspartyl proteinase enzyme activity in-vitro. Biofilm forming, drug-resistant clinical isolates of C. albicans (n = 25) and non-albicans (n= 20) were assessed for their ability to reduce the metabolic and aspartyl proteinase activities using XTT assay and spectrophotometric analysis at different concentrations of mycogenic Ag NPs. After 24 h of incubation, significant reduction (>50%) in metabolic activity was observed with 100 ppm mycogenic Ag NPs. Incubation time has greater inhibitory effect against Candida spp. biofilms secreted aspartyl proteinase after treatment with 100 ppm mycogenic Ag NPs. Inhibition of secreted aspartyl proteinase by mycogenic Ag NPs provides an insight towards the mechanism for the treatment of Candida-associated infections involving biofilms-related infections.

Outbreak of dengue virus type-3 in Malakand, Pakistan 2015; A laboratory perspective

An outbreak of dengue fever was reported in Malakand district, Khyber Pakhtunkhwa (KP) province of Pakistan during 2015. Detection of viral RNA by real-time PCR confirmed dengue virus serotype-3 (DENV-3) to be the causative agent causing the outbreak. Phylogenetic analysis based on partial E-NS1 gene sequences showed that the DENV-3 viruses belonged to genotype III with maximum homology with the dengue-3 strains previously reported from Pakistan and India. Our current report provides updated information on molecular epidemiology and phylogenetic analysis of dengue virus serotypes responsible for 2015 outbreak in KP.

Antibiotic Susceptibility and Molecular Characterization of Campylobacter jejuni Strain Isolated from a Guillain Barré Syndrome Child

To the Editor : Campylobacter jejuni is among the leading etiological agents associated with bacterial diarrhea worldwide. In rare occasions,Campylobacter jejuni infectionmay trigger the human immune system to attack self-gangliosides resulting in Guillain-Barré syndrome [1]. We here, present case of a 7-yold girl suffering from acute flaccid paralysis of lower limbs after an episode of bloody diarrhea. The child was wheel chair bounded but did not have any difficulty in breathing or swallowing and did not receive any antibiotic treatment. The patient showed elevated levels of cerebrospinal fluid (CSF) (261 csf mg/dl). Shewas treatedwith – 5d course of intravenous immunoglobulin (IVIG) at standard dose of 0.4 g/kg/d. No ventilation support was needed. On day 25th, she walked – 5m distance with aid. C. jejuni was isolated from the patient’s stool sample and identification was confirmed by using species specific (hipO) PCR [2]. The strain was found to be resistant to three antibiotics i.e., erythromycin, sulfamethoxazole + trimethoporim, cephalothin. PCR analysis for the detection of genes (sul1, sul2 and sul3) showed that the isolate was positive for all the three resistivity genes [3, 4]. Virulence genes i.e., cadF (fibronectin binding protein) and wlaN (putative beta-1,3-galactosyl transferase) were present whereas NeuAB (sialic acid biosynthesis gene) was absent [2]. ThewlaN gene inC. jejuni is proposed to encode an enzyme β-1,3-galactosyl transferase that converts GM2-like LOS structure to a GM1-like structure. These GM1-like structures trigger the human host to produce the anti-GM1 antibodies which binds to GM1 expressed on the motor nerves of the limbs, resulting in Guillain–Barré syndrome [5]. The presence of wlaN gene in the present isolate further confirms the role of the gene in triggering AMAN, a variant of GBS. To our knowledge this is the first case of GBS following infection with C. jejuni, reported from Pakistan. Further investigations on prevalence and molecular characterization of C. jejuni associated GBS may contribute to a better understanding of the disease.

NS1 antigen: A new beam of light in the early diagnosis of dengue infection

OBJECTIVE To evaluate NS1 antigen detection ELISA for the early laboratory diagnosis of dengue virus infection. METHODS The present study was conducted to evaluate the overall positivity of NS1 antigen detection ELISA and its comparison with viral RNA detection via real time PCR and IgM antibodies detection by ELISA. RESULTS A total of 1270 serum samples were tested 86% (1097/1270) were detected positive by one or more than one diagnostic test. Out of 1 270, 64% (807/1270) were positive by NS1 ELISA and 52% (662/1270), 51% (646/1270) were positive by real-time RT-PCR and IgM ELISA respectively. CONCLUSIONS NS1 antigen detection ELISA is highly suitable diagnostic tools and it also has great value for use in outbreak and epidemic situation.

Statin resistance in Candida glabrata

ObjectivesReduced efficacy of statins has been observed in people but the mechanism of this resistance is unclear and no statin-resistance mutations in the catalytic domain of HMGCR have been reported. The present study focused on looking for statin-resistance mutations and examining the mechanism of statin resistance using Candida glabrata as a model organism.ResultsC. glabrata was cultured in media containing lovastatin, simvastatin or atorvastatin to obtain lovastatin-, simvastatin- and atorvastatin-resistant mutants. A single mutant from each was purified for further analysis. In each mutant, gene sequencing showed there were no changes in the catalytic domain of HMGCR. HMGCR was overexpressed in two resistant isolates suggesting that increased production of HMGCR can lead to resistance. In a third mutant, HMGCR activity was unaltered, suggesting a non-HMGCR related mechanism, such as increased drug efflux, could be operating.ConclusionsCandida glabrata is a useful model organism for examining resistance to statins. Further studies are warranted to examine the precise molecular mechanisms of statin resistance.

Possible association of a distinct combined Glutathione-S-transferase members with allergic asthma patients in Pakistan

Allergic asthma is a diverse chronic respiratory disease characterized by the inflammation of the lower airway disease affecting many people around the world with rising morbidity and mortality. Association between asthma and certain demographic features was studied in relation to genotype from 244 allergic individuals of local population. Skin prick test was used to confirm asthma. Genetic polymorphism in Glutathione-S-transferases (GSTs) was studied using multiplex PCR based method and IgE level by ELISA. Pollen and dust were the major causative aeroallergens (26%), which were associated to higher IgE levels (P < 0.05). Smoking was found to be significantly associated with asthma in only males (P = 0.004). A low prevalence of null genotype of both GSTM1 and GSTT1 genes was observed in the patients (4.34%) compared to control group (14%). No association of combined GSTM1 and GSTT1 null genotype was found with the asthma in local population. GSTM1+ and GSTT− genotype had higher risk (OR = 1.3681, P = 0.001) for development of asthma. There was a significant association of asthma with combined genotype of GSTM1+ and GSTT− when data was analyzed on gender basis in males (P = 0.006) and highly significant in age range of 26–40 years (P = 0.001). Combined GSTM+ and GSTT− genotype was found to be risk factor for asthma in addition to family history in male patients. However a data with large patient size and different ethnic distribution may reveal the exact etiology.