Evaluating the Sensitivity of Different Molecular Techniques for Detecting Mycobacterium tuberculosis Complex in Patients with Pulmonary Infection.

This study aimed to evaluate the accuracy of detecting drug-resistant Mycobacterium tuberculosis complex (MTBC)-specific DNA in sputum specimens from 48 patients diagnosed with pulmonary tuberculosis. The presence of MTBC DNA in the specimens was validated using the GeneXpert MTB/RIF system and compared with a specific PCR assay targeting the IS6110 and the mtp40 gene sequence fragments. Additionally, the results obtained by multiplex PCR assays to detect the most frequently encountered rifampin, isoniazid, and ethambutol resistance-conferring mutations were matched with those obtained by GeneXpert and phenotypic culture-based drug susceptibility tests. Of the 48 sputum samples, 25 were positive for MTBC using the GeneXpert MTB/RIF test. Nevertheless, the IS6110 and mtp40 single-step PCR revealed the IS6110 in 27 of the 48 sputum samples, while the mtp40 gene fragment was found in only 17 of them. Furthermore, multiplex PCR assays detected drug-resistant conferring mutations in 21 (77.8%) of the 27 samples with confirmed MTBC DNA, 10 of which contained single drug-resistant conferring mutations towards ethambutol and two towards rifampin, and the remaining nine contained double-resistant mutations for ethambutol and rifampin. In contrast, only five sputum specimens (18.5%) contained drug-resistant MTBC isolates, and two contained mono-drug-resistant MTBC species toward ethambutol and rifampin, respectively, and the remaining three were designated as multi-drug resistant toward both drugs using GeneXpert and phenotypic culture-based drug susceptibility tests. Such discrepancies in the results emphasize the need to develop novel molecular tests that associate with phenotypic non-DNA-based assays to improve the detection of drug-resistant isolates in clinical specimens in future studies.

Blood culture contamination in a tertiary care hospital of Saudi Arabia. A one-year study.

OBJECTIVES: To monitor blood culture contamination (BCC) rates in a tertiary care hospital in Saudi Arabia. Methods: Blood cultures submitted to the Microbiology Laboratory of King Fahad Hospital, Madina, Saudi Arabia between January and December 2017 were analyzed prospectively. Positive blood cultures were either designated as true bacteremia with confirmed bloodstream infection or BCC. RESULTS: Among 5,536 blood cultures from 2201 patients, 364 (6.6%) mirrored BCC. There was an upward trend in contamination rates in specific months. With respect to total blood cultures from respective units over a one-year period, medical ward contributed to the highest contamination rate (10.3%). Blood culture contamination rate in the wards ranged from 4.5-10.3%, with a higher contamination rate in elderly, aged 60-80 years. Staphylococcus epidermidis (S. epidermidis) was the most frequent contaminant (44.5%). Conclusion: The escalated contamination rates in September to October may be attributed to difficulty in sampling blood by the less competent nurses during annual pilgrimage season. High influx of patients and shortage of trained nurses may have resulted in increased incidence in December-January and March-April. The prevalence of skin-resident S. epidermidis may be due to improper aseptic conditions. Ours is the first report on evaluation of BCC rates in Madina and call for renewed efforts in this direction.

Cinnamomum cassia exhibits antileishmanial activity against Leishmania donovani infection in vitro and in vivo.

BACKGROUND: There is a pressing need for drug discovery against visceral leishmaniasis, a life-threatening protozoal infection, as the available chemotherapy is antiquated and not bereft of side effects. Plants as alternate drug resources has rewarded mankind in the past and aimed in this direction, we investigated the antileishmanial potential of Cinnamomum cassia. METHODOLOGY: Dichloromethane, ethanolic and aqueous fractions of C. cassia bark, prepared by sequential extraction, were appraised for their anti-promastigote activity along with apoptosis-inducing potential. The most potent, C. cassia dichloromethane fraction (CBD) was evaluated for anti-amastigote efficacy in infected macrophages and nitric oxide (NO) production studied. The in vivo antileishmanial efficacy was assessed in L. donovani infected BALB/c mice and hamsters and various correlates of host protective immunity ascertained. Toxicity profile of CBD was investigated in vitro against peritoneal macrophages and in vivo via alterations in liver and kidney functions. The plant secondary metabolites present in CBD were identified by gas chromatography-mass spectroscopy (GC-MS). PRINCIPAL FINDINGS: CBD displayed significant anti-promastigote activity with 50% inhibitory concentration (IC50) of 33.6 µg ml-1 that was mediated via apoptosis. This was evidenced by mitochondrial membrane depolarization, increased proportion of cells in sub-G0-G1 phase, ROS production, PS externalization and DNA fragmentation (TUNEL assay). CBD also inhibited intracellular amastigote proliferation (IC50 14.06 µg ml-1) independent of NO production. The in vivo protection achieved was 80.91% (liver) and 82.92% (spleen) in mice and 75.61% (liver) and 78.93% (spleen) in hamsters indicating its profound therapeutic efficacy. CBD exhibited direct antileishmanial activity, as it did not specifically induce a T helper type (Th)-1-polarized mileu in cured hosts. This was evidenced by insignificant modulation of NO production, lymphoproliferation, DTH (delayed type hypersensitivity), serum IgG2a and IgG1 levels and production of Th2 cytokines (IL-4 and IL-10) along with restoration of pro-inflammatory Th1 cytokines (INF-γ, IL-12p70) to the normal range. CBD was devoid of any toxicity in vitro as well as in vivo. The chemical constituents, cinnamaldehyde and its derivatives present in CBD may have imparted the observed antileishmanial effect. CONCLUSIONS: Our study highlights the profound antileishmanial efficacy of C. cassia bark DCM fraction and merits its further exploration as a source of safe and effective antieishmanial compounds.

Nanoliposomal artemisinin for the treatment of murine visceral leishmaniasis.

Visceral leishmaniasis (VL) is a fatal, vector-borne disease caused by the intracellular protozoa of the genus Leishmania. Most of the therapeutics for VL are toxic, expensive, or ineffective. Sesquiterpenes are a new class of drugs with proven antimicrobial and antiviral activities. Artemisinin is a sesquiterpene lactone with potent antileishmanial activity, but with limited access to infected cells, being a highly lipophilic molecule. Association of artemisinin with liposome is a desirable strategy to circumvent the problem of poor accessibility, thereby improving its efficacy, as demonstrated in a murine model of experimental VL. Nanoliposomal artemisinin (NLA) was prepared by thin-film hydration method and optimized using Box-Behnken design with a mean particle diameter of 83±16 nm, polydispersity index of 0.2±0.03, zeta potential of -27.4±5.7 mV, and drug loading of 33.2%±2.1%. Morphological study of these nanoliposomes by microscopy showed a smooth and spherical surface. The mechanism of release of artemisinin from the liposomes followed the Higuchi model in vitro. NLA was free from concomitant signs of toxicity, both ex vivo in murine macrophages and in vivo in healthy BALB/c mice. NLA significantly denigrated the intracellular infection of Leishmania donovani amastigotes and the number of infected macrophages ex vivo with an IC50 of 6.0±1.4 µg/mL and 5.1±0.9 µg/mL, respectively. Following treatment in a murine model of VL, NLA demonstrated superior efficacy compared to artemisinin with a percentage inhibition of 82.4%±3.8% in the liver and 77.6%±5.5% in spleen at the highest dose of 20 mg/kg body weight with modulation of cell-mediated immunity towards protective Th1 type. This study is the first report on the use of a liposomal drug delivery system for artemisinin as a promising alternative intervention against VL.

Epidemiological Trends of Cutaneous Leishmaniasis in Al-Madinah Al-Munawarah Province, Western Region of Saudi Arabia.

OBJECTIVE: To investigate the epidemiological trends of cutaneous leishmaniasis (CL) in Al-Madinah Al-Munawarah, western region of KSA. MATERIALS AND METHODS: Four hundred and sixty-seven parasitologically confirmed CL cases attending Al-Meeqat Hospital, Al-Madinah, during 2012-2015, were included in this study. RESULTS: Both Saudi and non-Saudi nationals were infected, with the highest infection rate being among Saudis (68.7%). Males were more affected than females as 86.9% of the total CL cases were males. Moreover, CL was prevalent in all age groups with higher frequency among young adults and adolescents (23.1% and 22.7%, respectively). Interestingly, almost all the patients in the adolescent and child age groups were Saudis (96.2% and 93.5%, respectively). Considering geographical distribution, the highest percentage of the cases (40.5%) were from the northern parts of Al-Madinah province while the eastern parts reported the least infection rate (7.3%). Few cases (2.5%) were supposed to encounter the infection abroad. Additionally, the frequency of infection was found to follow a seasonal distribution. Regarding treatment, pentostam, ketoconazole, or cryotherapy were the treatment options usually used. CONCLUSION: CL is prevalent in Al-Madinah Al-Munawarah area and new foci are being introduced. Thus, detailed studies with large surveillances regarding vector and reservoir hosts in and around the area are needed.

Immunotherapeutic Potential of Eugenol Emulsion in Experimental Visceral Leishmaniasis.

BACKGROUND: The therapy of visceral leishmaniasis (VL) is limited by resistance, toxicity and decreased bioavailability of the existing drugs coupled with dramatic increase in HIV-co-infection, non-availability of vaccines and down regulation of cell-mediated immunity (CMI). Thus, we envisaged combating the problem with plant-derived antileishmanial drug that could concomitantly mitigate the immune suppression of the infected hosts. Several plant-derived compounds have been found to exert leishmanicidal activity via immunomodulation. In this direction, we investigated the antileishmanial activity of eugenol emulsion (EE), complemented with its immunomodulatory and therapeutic efficacy in murine model of VL. METHODOLOGY/PRINCIPAL FINDINGS: Oil-in-water emulsion of eugenol (EE) was prepared and size measured by dynamic light scattering (DLS). EE exhibited significant leishmanicidal activity with 50% inhibitory concentration of 8.43±0.96 µg ml-1 and 5.05±1.72 µg ml─1, respectively against the promastigotes and intracellular amastigotes of Leishmania donovani. For in vivo effectiveness, EE was administered intraperitoneally (25, 50 and 75 mg/kg b.w./day for 10 days) to 8 week-infected BALB/c mice. The cytotoxicity of EE was assessed in RAW 264.7 macrophages as well as in naive mice. EE induced a significant drop in hepatic and splenic parasite burdens as well as diminution in spleen and liver weights 10 days post-treatment, with augmentation of 24h-delayed type hypersensitivity (DTH) response and high IgG2a:IgG1, mirroring induction of CMI. Enhanced IFN-γ and IL-2 levels, with fall in disease-associated Th2 cytokines (IL-4 and IL-10) detected by flow cytometric bead-based array, substantiated the Th1 immune signature. Lymphoproliferation and nitric oxide release were significantly elevated upon antigen revoke in vitro. The immune-stimulatory activity of EE was further corroborated by expansion of IFN-γ producing CD4+ and CD8+ splenic T lymphocytes and up-regulation of CD80 and CD86 on peritoneal macrophages. EE treated groups exhibited induction of CD8+ central memory T cells as evidenced from CD62L and CD44 expression. No biochemical alterations in hepatic and renal enzymes were observed. CONCLUSIONS: Our results demonstrate antileishmanial activity of EE, potentiated by Th1 immunostimulation without adverse side effects. The Th1 immune polarizing effect may help to alleviate the depressed CMI and hence complement the leishmanicidal activity.

Corrigendum: Leishmanicidal Activity of Piper nigrum Bioactive Fractions is Interceded via Apoptosis In Vitro and Substantiated by Th1 Immunostimulatory Potential In Vivo.

[This corrects the article on p. 1368 in vol. 6, PMID: 26696979.].

Elevated TGF-beta levels in drug-resistant visceral leishmaniasis.

BACKGROUND: Poor and neglected populations in Africa are particularly affected with visceral leishmaniasis. The widespread emergence of resistance to pentavalent antimonials occurs globally and the unavailability of a vaccine in clinical use constitutes a major obstacle in disease control. OBJECTIVE: To investigate the cytokine profile in human visceral leishmaniasis. DESIGN: A cross-sectional laboratory-based study. SETTING: Single center study carried out at the Institute of Endemic Diseases, University of Khartoum, Sudan. PATIENTS AND METHODS: Soluble lysates of L major and L donovani were used to stimulate the lymphocytes of two groups of confirmed VL patients (group 1 [n=20] had respond to pentostam treatment and group 2 [n=5] were recorded as drug resistant after follow up) in a cellular proliferation assay and the levels of IFNg, IL-10, TNFa and TGFb were detected by cytokine ELISA. MAIN OUTCOME MEASURES: Levels of IFNg, TNFa, IL-10 and TGFb. RESULTS: A significant increase of IFNg and TNFa levels were reported in stimulated cells of drug susceptible and drug resistant groups, but no significant difference in IL-10 production was observed between the different antigens or between the patients groups. TGFb from stimulated lymphocytes was secreted in statistically significant amounts in patients reported as drug resistant in response to both L major and L donovani antigens (P < .001). CONCLUSIONS: In VL patients, IFNg and TNFa are extremely produced in response to in vitro re-stimulation which means that the parasitic infection, although virulent and chronic, does not render patients as immunocompromised. However, TGFb is mostly associated with treatment failure. LIMITATIONS: This study assessed secretory TGFb. A study with a larger sample size to assess TGFb gene expression and to follow its intracytoplasmic synthesis in drug resistant VL patients is recommended.

Leishmanicidal Activity of Piper nigrum Bioactive Fractions is Interceded via Apoptosis In Vitro and Substantiated by Th1 Immunostimulatory Potential In Vivo.

Visceral leishmaniasis (VL) is a life-threatening protozoal infection chiefly impinging the rural and poor population in the tropical and sub-tropical countries. The deadly affliction is rapidly expanding after its association with AIDS, swiftly defying its status of a neglected disease. Despite successful formulation of vaccine against canine leishmaniasis, no licensed vaccine is yet available for human VL, chemotherapy is in appalling state, and the development of new candidate drugs has been painfully slow. In face of lack of proper incentives, immunostimulatory plant preparations owing antileishmanial efficacy bear potential to rejuvenate awful antileishmanial chemotherapy. We have earlier reported profound leishmanicidal activity of Piper nigrum hexane (PNH) seeds and P. nigrum ethanolic (PNE) fractions derived from P. nigrum seeds against Leishmania donovani promastigotes and amastigotes. In the present study, we illustrate that the remarkable anti-promastigote activity exhibited by PNH and PNE is mediated via apoptosis as evidenced by phosphatidylserine externalization, DNA fragmentation, arrest in sub G0/G1 phase, loss of mitochondrial membrane potential and generation of reactive oxygen species. Further, P. nigrum bioactive fractions rendered significant protection to L. donovani infected BALB/c mice in comparison to piperine, a known compound present in Piper species. The substantial therapeutic potential of PNH and PNE was accompanied by elicitation of cell-mediated immune response. The bioactive fractions elevated the secretion of Th1 (INF-γ, TNF-α, and IL-2) cytokines and declined IL-4 and IL-10. PNH and PNE enhanced the production of IgG2a, upregulated the expression of co-stimulatory molecules CD80 and CD86, augmented splenic CD4(+) and CD8(+) T cell population, induced strong lymphoproliferative and DTH responses and partially stimulated NO production. PNH and PNE were devoid of any hepatic or renal toxicity. These encouraging findings merit further exploration of P. nigrum bioactive fractions as a source of potent and non-toxic antileishmanials.

Absence of the mecA Gene in Methicillin Resistant Staphylococcus aureus Isolated from Different Clinical Specimens in Shendi City, Sudan.

Absolute dependence on mecA gene as the defining standard in determining the resistance of S. aureus to methicillin became the subject of distrust by many researchers. The present study aimed to determine the frequency of mecA gene in methicillin resistant S. aureus (MRSA) isolates using polymerase chain reaction and to correlate its presence to conventional method. In this regard, two hundred S. aureus isolates were collected from patients with different diseases attending different hospitals in Shandi City, Sudan. Phenotypic Kirby-Bauer method confirmed the existence of methicillin resistant S. aureus in 61.5% of the subjected isolates with MICs ranging from 4 µg/mL to 256 µg/mL when using E-test. However, when amplifying a 310 bp fragment of the mecA gene by PCR, twelve out of the 123 MRSA isolates (9.8%) were mecA negative, whereas all the 77 methicillin sensitive S. aureus (MSSA) were mecA negative. In conclusion, this study drew attention to the credibility of the mecA gene and its usefulness in the detection of all MRSA strains without referring to the traditional methods. Hence, it is highly recommended to consider alternative mechanisms for ß-lactam resistance that may compete with mecA gene in the emergence of MRSA phenomenon in the community.

Effect of essential oil of traditional two Saudi mint types and its possible role in cardiovascular and throat health.

Essential Oil (EO) of two Mentha species (popularly used in Saudi Arabia), Mentha Longifolia (ML), and Mentha pulegium (MP) was isolated and subjected to inhibit LDL oxidation in 22 hypercholestrolemic samples collected from 22 subjects, and inhibited of 5 bacterial pathogen in vitro. LDL was isolated by ultracentrifugation and enhanced to oxidation with CuSO4 and EO was added to prevent this oxidation, free radical scavenging activity was measured (decrease 50% DPPH radicals). EO content of phenolic and flavonoid was estimated. Five bacterial strains infect human throat was tested against EO of ML and MP in diffusion agar method. EO of the two herbals possess highly significantresults, success to inhibit LDL oxidation (P<0.001 for both herbal than non addition against lipid peroxidase, P<0.001 ML better than MP P<0.001), decreased DPPH free radicals (P<0.001 for both than control, P<0.001 for ML than MP), and possess strong antioxidant activity for ML than MP (polyphenol and flavonoids contents was higher in EO of ML than MP, P<0.001). EO of ML possess strong antioxidant and antibacterial activity than MP, these attributed to its high flavonoid contents enable it to be a good for cardiovascular and throat health. The study supported the traditional uses of ML.

Therapeutic efficacy of artemisinin-loaded nanoparticles in experimental visceral leishmaniasis.

Visceral leishmaniasis (VL) is a fatal vector-borne parasitic syndrome attributable to the protozoa of the Leishmania donovani complex. The available chemotherapeutic options are not ideal due to their potential toxicity, high cost and prolonged treatment schedule. In the present study, we conjectured the use of nano drug delivery systems for plant-derived secondary metabolite; artemisinin as an alternative strategy for the treatment of experimental VL. Artemisinin-loaded poly lactic co-glycolic acid (ALPLGA) nanoparticles prepared were spherical in shape with a particle size of 220.0±15.0 nm, 29.2±2.0% drug loading and 69.0±3.3% encapsulation efficiency. ALPLGA nanoparticles administered at doses of 10 and 20mg/kg body weight showed superior antileishmanial efficacy compared with free artemisinin in BALB/c model of VL. There was a significant reduction in hepatosplenomegaly as well as in parasite load in the liver (85.0±5.4%) and spleen (82.0±2.4%) with ALPLGA nanoparticles treatment at 20mg/kg body weight compared to free artemisinin (70.3±0.6% in liver and 62.7±3.7% in spleen). In addition, ALPLGA nanoparticle treatment restored the defective host immune response in mice with established VL infection. The protection was associated with a Th1-biased immune response as evident from a positive delayed-type hypersensitivity reaction, escalated IgG2a levels, augmented lymphoproliferation and enhancement in proinflammatory cytokines (IFN-γ and IL-2) with significant suppression of Th2 cytokines (IL-10 and IL-4) after in vitro recall, compared to infected control and free artemisinin treatment. In conclusion, our results advocate superior efficacy of ALPLGA nanoparticles over free artemisinin, which was coupled with restoration of suppressed cell-mediated immunity in animal models of VL.

Apoptosis mediated leishmanicidal activity of Azadirachta indica bioactive fractions is accompanied by Th1 immunostimulatory potential and therapeutic cure in vivo.

BACKGROUND: Exploration of immunomodulatory antileishmanials of plant origin is now being strongly recommended to overcome the immune suppression evident during visceral leishmaniasis (VL) and high cost and toxicity associated with conventional chemotherapeutics. In accordance, we assessed the in vitro and in vivo antileishmanial and immunomodulatory potential of ethanolic fractions of Azadirachta indica leaves (ALE) and seeds (ASE). METHODS: A. indica fractions were prepared by sequential extraction of the powdered plant parts in hexane, ethanol and water. Erythrosin B staining was employed to appraise the anti-promastigote potential of ALE and ASE. Cytostatic or cytocidal mode of action was ascertained and alterations in parasite morphology were depicted under oil immersion light microscopy. Study of apoptotic correlates was performed to deduce the mechanism of induced cell death and anti-amastigote potential was assessed in Leishmania parasitized RAW 264.7 macrophages. In vivo antileishmanial effectiveness was evaluated in L. donovani infected BALB/c mice, accompanied by investigation of immunomodulatory potential of ALE and ASE. Adverse toxicity of the bioactive fractions against RAW macrophages was studied by MTT assay. In vivo side effects on the liver and kidney functions were also determined. Plant secondary metabolites present in ALE and ASE were analysed by Gas chromatography-mass spectrometry (GC-MS). RESULTS: ALE and ASE (500 µg ml(-1)) exhibited leishmanicidal activity in a time- and dose-dependent manner (IC50 34 and 77.66 µg ml(-1), respectively) with alterations in promastigote morphology and induction of apoptosis. ALE and ASE exerted appreciable anti-amastigote potency (IC50 17.66 and 24.66 µg ml(-1), respectively) that was coupled with profound in vivo therapeutic efficacy (87.76% and 85.54% protection in liver and 85.55% and 83.62% in spleen, respectively). ALE exhibited minimal toxicity with selectivity index of 26.10 whereas ASE was observed to be non-toxic. The bioactive fractions revealed no hepato- and nephro-toxicity. ALE and ASE potentiated Th1-biased cell-mediated immunity along with upregulation of INF-γ, TNF-α and IL-2 and decline in IL-4 and IL-10 levels. GC-MS analysis revealed several compounds that may have contributed to the observed antileishmanial effect. CONCLUSION: Dual antileishmanial and immunostimulatory efficacy exhibited by the bioactive fractions merits their use alone or as adjunct therapy for VL.

Influence of lipoic acid on testicular toxicity induced by bi-n-butyl phthalate in rats.

Bi-n-butyl phthalate (BNBP) is an environmental pollutant. The aim of this study was to evaluate the protective effect of lipoic acid (LA) against testicular dysfunction associated with the intake of to BNBP- intoxicated rats. Adult male Wistar rats were divided into 4 groups of 6 animals each, and received medication orally for 14 days. Group I rats received 0.5 ml corn oil. Group II rats received LA (20 mg/kg B.W./day). Group III rats received BNBP (250 mg/kg B.W./day). Group IV rats received LA 24h prior to BNBP intake. Testes weight, cauda sperm count and sperm motility were decreased significantly by 18.15%, 13.83% and 13.5%, respectively, after BNBP treatment. Significant increase by 12.1%, 10.20% and 11.51%, respectively, was observed in LA-BNBP rats. Significant increase by 1.53%, 1.5% and 1.8%, for serum follicle stimulating hormone, testosterone and total antioxidant status, respectively, were observed in LA-BNBP rats. Testicular lipid peroxides and lactate dehydrogenase enzyme were significantly decreased by 1.5 and 1.6 folds, respectively, in LA-BNBP rats were decreased after BNBP treatment. Testicular superoxide dismutase, catalase and glutathione reductase enzymes were significantly increased in LA-BNBP rats. LA-BNBP rats, decreased the damage to seminiferous tubules produced by BNBP intake. In conclusion, LA mitigated BNBP-induced testicular toxicity through antioxidant mechanism and by direct free radical scavenging activity.