Pulmonary nocardiosis caused by Nocardia otitidiscaviarum in an adult asthmatic female patient: The presence of acid-fast branching filaments is always significant.

We report a case of pulmonary nocardiosis in a 41-year-old asthma patient. Chest radiographs showed bilateral air space and consolidations.Acid-fast branching filaments were demonstrated in sputum, and the grown organism was identified phenotypically and confirmed using16S rDNA sequencing (accession no. KX500116). The patient received a combination of medical treatments, but developed complications,which were managed over the next 3 months, after which she was discharged. Pulmonary nocardiosis should be considered in patientsundergoing steroid therapy or when a chronic infection does not respond to first-line treatment.

Chronic rhinofacial basidiobolomycosis caused by Basidiobolus ranarum: Report of a case from Aseer Region, Kingdom of Saudi Arabia.

We present a case of fungal sinusitis caused by Basidiobolus ranarum in a 22-year-old male patient with chronic rhinosinusitis in Aseer region, Kingdom of Saudi Arabia. The patient was admitted with nasal obstruction accompanied by itching, sneezing, rhinorrhea, epistaxis and recurrent headache. Axial computed tomography (CT) scan of the paranasal sinuses showed a clear left facial swelling chronic inflammation and granulomata. Basidiobolus ranarum fungus was isolated on Sabouraud dextrose agar from a biopsy specimen. The organism was characterized by flat, yellowish-grey, glabrous, becoming radially folded fungus that under the microscope showed broad vegetative hyaline hyphae that bear zygospores with protuberances. The patient made good recovery and was discharged home with no recurrences after receiving oral itraconazole and removal of the polyps surgically.

In vitro anti-yeast activity of chloramphenicol: A preliminary report.

Chloramphenicol is a bacteriostatic antimicrobial agent but its antifungal activity is not known. The present study aimed to investigate the activity of chloramphenicol against 30 representative yeasts. The antimicrobial assay of chloramphenicol (50mg/mL; 100mg/mL and 200mg/mL) was determined by the disc diffusion method using Mueller-Hinton agar against 30 representative yeast strains. Zone of inhibition was read after 48-72h incubation at 37°C and results were compared with some standard antifungal agents. Most of the tested yeasts (73.3%) showed inhibition zones (5 up to 35mm) to chloramphenicol impregnated discs (200mg/mL). Three out of the four tested Candida albicans as well as Candida famata, Candida glabrata, Candida haemolonei and Cryptococcus neoformans showed no inhibition zones to chloramphenicol (200mg/mL). Caspofungin acetate (50mg/mL) inhibited 83.3% of the strains; ketoconazole (200mg/mL) 70% and metronidazole 10%. Chloramphenicol discs: 50 and 100mg/mL showed less activity (6.7% and 36.7%, respectively) compared to the 200mg discs; whereas chloramphenicol (BBL; 3µg/mL) inhibited 13.3% of the strains. The anti-yeast activities of chloramphenicol were comparable to other known antifungal compounds. Moreover, it is cheap, has fewer side effects and its inclusions in selective fungal media such as Mycosel have to be questioned.

Molecular identification of nontuberculous mycobacteria isolated from pyogenic bovine tissues in South Darfur State and Alsabalouga slaughterhouse at Omdurman area, Sudan.

This study identified nontuberculous mycobacteria (NTM) recovered from bovine pyogenic affections obtained at necropsy using the molecular target 16S-23S rDNA internal transcribed spacer region. Postmortem inspection of cattle was conducted at South Darfur State abattoirs and Alsabalouga Slaughterhouse at Omdurman area during 2007-2009. Specimens were examined for the presence of acid fast bacteria (AFB) using microscopic and standard culturing techniques. AFB were identified phenotypically and confirmed by 16S-23S rDNA ITS. Fifty nine NTM were recovered and confirmed as acid fast filaments out of 165 positive AFB specimens, of which 52 isolates were identified as bovine farcy causative agents, while 7 cultures were excluded due to drying. 16S-23S rDNA ITS of NTM revealed three different amplicons 500 bp. (32) isolates, 550 bp. (2) isolates and 600 bp. (14) isolates. Four isolates were contaminated.

Increased multi-drug resistant Escherichia coli from hospitals in Khartoum state, Sudan.

BACKGROUND: Multidrug-resistant Escherichia coli (MDR E. coli) has become a major public health concern in Sudan and many countries, causing failure in treatment with consequent huge health burden. OBJECTIVES: To determine the prevalence and susceptibility of MDR E. coli isolated from patients in hospitals at Khartoum State. METHODS: Between May to August 2011, E. coli (n = 232) isolated from clinical specimens, identified, tested their antimicrobials susceptibility and screened for extend spectrum â-lactamase production as per standard methods. RESULTS: Of the 232 E. coli isolates, the majority were from urine (65.1%). MDR E. coli were present in 214 (92.2%). Of these, the resistance rates were recorded to: amoxicillin 97.7%, cefuroxime 92.5%, trimethoprim-sulfamethoxazole 88.3%, tetracycline 77.1%, nalidixic acid 72%, ceftriaxone 64%, ciprofloxacin 58.4%, ofloxacin 55.1%, amoxicillin-clavulanate 50.4%, ceftazidime, gentamicin 35% each, nitrofurantoin 22.4%, chloramphenicol, tobramicin 18.2% each and amikacin 1.9%. Overall MDR E. coli, 53.3% were resistant to > 7 antimicrobial agents and ESBL was detected in 32.7%. Isolates from males were more resistant than those from females (p < 0.05). CONCLUSIONS: Drug-resistance surveillance and epidemiological analysis of patient data is need periodically and can be informative for appropriate management of antimicrobial resistance.

Evaluation of ELISA in the serodiagnosis of bovine farcy.

Mycobacterium farcinogenes is the causal agent of bovine farcy, a chronic infectious disease of zebu cattle in some parts of tropical Africa. Whole cell homologous antigen of M. farcinogenes was used in the standardization and evaluation of an enzyme linked immunosorbent assay (ELISA) for the detection of circulating antibodies against bovine farcy using sera from confirmed bovine farcy and from bovine farcy-free cattle. The cut-off optical density (OD) value was decided at 1.8 using filter 405 nm after one hour of incubation at 37 degrees C. Accordingly, 115 out of 124 (92.7%) serum samples from clinically proven bovine farcy cattle were reported sero-positive. Sera from cattle infected with M. avium and M. paratuberculosis revealed OD value <1.8, indicating the differential diagnostic ability of M. farcinogenes antigen. Our test sensitivity was 92.7% and specificity was 97%, therefore could be routinely employed to support early clinical diagnosis, epidemiological surveys and for screening animals before exportation to farcy-free regions.

The treatment of bovine dermatophilosis and its effect on some haematological and blood chemical parameters.

In this study, the authors evaluated parenteral treatment of zebu cattle, with naturally and experimentally induced bovine dermatophilosis, in western Sudan, using four different antibiotic treatments. In terms of recovery rate, weight gain, avoiding relapse and preventing death, gentamycin was found to be the most effective treatment, followed by a combination of penicillin and streptomycin and, finally, long-acting oxytetracycline. However, enrofloxacin was not successful. A significant improvement in the red blood cell count was noticed among cattle treated with penicillin-streptomycin (p = 0.021) and gentamycin (p = 0.029). All treated cattle, except those treated with enrofloxacin, showed a significant improvement in mean corpuscular haemoglobin concentration (p = 0.021); mean corpuscular volume (p = 0.021), and white blood cell count (p < 0.021). Significant improvements were observed among treated cattle in their total levels of protein, calcium (p = 0.021) and cholesterol (p < 0.05), when compared to untreated cattle infected with Dermatophilus congolensis. This study recommends gentamycin as a drug of choice for the parenteral treatment of dermatophilosis. Treatment was not only effective in early, mild cases but also useful among moderately and heavily affected cattle. According to the observations of the authors, when no intervention took place, the condition of moderately and heavily affected cattle deteriorated and/or resulted in death.

Comparative assays of the rpoB gene for identification of Mycobacterium tuberculosis isolated from patients in Sudan.

OBJECTIVES: To characterise mycobacterial clinical isolates based on amplification of the rpoB gene. SETTING: One hundred and thirty-five mycobacterial isolates cultured from suspected pulmonary tuberculosis (TB) patients were identified phenotypically. Molecular characterisation of the isolates was performed based on amplification of the rpoB gene, using duplex polymerase chain reaction (DPCR), PCR-restriction fragment length polymorphism (RFLP) and nested PCR-based sequence analysis techniques. RESULTS: The DPCR assay identified 129 of 135 (95.5%) clinical isolates as Mycobacterium tuberculosis complex species. Restriction enzyme analysis of the rpoB PCR product using Hind II identified 134 of the 135 (99.3%) isolates as M. tuberculosis complex, while nested PCR sequence analysis of the rpoB gene identified 133/133 examined isolates (100%) as M. tuberculosis species. No mycobacteria other than M. tuberculosis (MOTT) were detected among the studied isolates. CONCLUSION: DPCR, PCR/RFLP Hind II and nested PCR sequence analysis of the rpoB gene techniques showed comparable efficiency in the characterisation of Mycobacterium isolates. Nested PCR sequence analysis of the rpoB gene was superior to PCR/RFLP for characterisation of suspected M. tuberculosis isolates, while the DPCR technique showed less sensitivity. As PCR-RFLP requires less sophisticated laboratory facilities than nested PCR sequence analysis, it would be more appropriate to be adopted for accurate characterisation of mycobacteria in countries with a weak infrastructure.

Unusual manifestation of a concurrent demodectic and sarcoptic mange in a Zebu-Friesian cross-bred heifer.

We describe a rare case of a concurrent demodectic and sarcoptic mange in a 2-year-old heifer in Khartoum, Sudan. The lesions were massive lumps of granulomatous tumour-like dermatitis with thick, nodular folds mainly covering the head, neck and shoulders. Histopathological examination of the lesions revealed the presence of both Demodex bovis and Sarcoptes scabiei. The animal died regardless of the anti-parasitic treatment it received.

Nocardia farcinica--a significant cause of mastitis in goats in Sudan.

Fifteen of 100 mastitic milk samples from goats suffering from mastitis were tentatively identified as members of the genus Nocardia on the basis of selected phenotypic and chemotaxonomic characteristics. Six of the 155 strains were confirmed as Nocardia farcinica by 16S rDNA gene sequencing and subsequent aligning with relevant actinomycetes found in electronic databases and 2 by other identification criteria. N. farcinica is a serious cause of mastitis with a significant prevalence (15%) among the examined goats. Efforts are needed to optimise and simplify isolation and identification methods.

Identification of acid fast bacteria from caseous lesions in cattle in Sudan.

One-hundred-and-twenty caseous lesions collected from slaughtered cattle at selected slaughterhouses in Sudan were processed for the detection of acid-fast bacteria (AFB). Sixty-four of the 120 samples showed AFB on microscopic examination after staining with the Ziehl-Neelsen method. Accordingly, it was estimated that 64 (53.3%) of the 120 caseous (purulent) lesions among the samples were due to AFB whereas 56 (46.7%) were due to other causes. Growth on Lowenstein-Jensen slants was obtained in 54 of the 120 samples. The isolated AFB were tentatively identified using microscopic and cultural characteristics. Confirmation of the phenotypic clusters was achieved by analysing the mycolic acids contents and PCR-amplification of the IS6110 insertion sequences. The above two methods have allowed the identification of Mycobacterium bovis and M. farcinogenes, the major AFB isolated from cattle in Sudan. The remaining AFB, which were negative for the above two tests, were further identified by sequencing the 16S rRNA gene. The above strategy thus allowed the identification of the isolated strains as follows: 25 (46%) M. bovis; 21 (39.9%) M. farcinogenes; 4 (7.4%) M. tuberculosis; 1 (1.9%) M. avium; 1 (1.9%) Nocardia sp., 2 (3.7%) unidentified AFB. The isolation of M. farcinogenes and M. tuberculosis, from pulmonary lymph nodes represented important findings.

Streptomyces sp., a cause of fistulous withers in donkeys.

Four of 10 donkeys, which showed lesions simulating fistulous withers, were examined clinically with the aim to cultivate and identify the causal agent. Aspiated purulent materials were subjected to bacteriological examination. The causal organisms were recovered in Tryptic Soya agar medium when incubated aerobically at 37 degrees C for up to 5 days. These organisms were found to be actinomycetes-like, Gam positive with stable branching filaments and to form heavy aerial hyphae on colony surface. The isolated organisms ere tentatively identified as Streptomyces sp. on the basis of morphological and cultural characteristics. The initial sequences analysis of the 16S rDNA gene conformed that one of the isolates (SD551) falls within the phylogenetic clade, which encompasses the genus Streptomyces. Studies are underway to further describe the disease and its causal agent. The report represents a good evidence to incriminate Streptomyces in the aetiology of the fistulous withers.

Molecular analysis of clinical isolates of Mycobacterium tuberculosis collected from patients with persistent disease in the Khartoum region of Sudan.

SETTING: Patients with positive smears for acid-fast bacilli were enrolled at tuberculosis (TB) clinics in the Khartoum region of Sudan. OBJECTIVE: To identify the presence of drug resistant genotypes in M. tuberculosis isolates which are difficult to treat. METHODS: Genus specific PCR-SSCP was performed to confirm the presence of M. tuberculosis in clinical isolates. Genotypic drug resistance testing was performed by mutation analysis and spoligotyping was used to monitor transmission and to identify epidemic strains. RESULTS: Fifty (48%) of the original 105 samples were classified as M. tuberculosis. Four (4%) of the samples were typed as mycobacteria other than TB, while the remaining (n =50) samples were refractory to further molecular analysis. The fifty amplifiable M. tuberculosis samples were used for subsequent mutation analysis and typing. Mutations were identified in the genes conferring resistance to INH (kat G, 12%), RIF (rpoB, 8%), SM (r psL and rrs, 30%) and EMB (embB, 4%). Two of the samples (4%) had mutations in genes associated to both INH and RIF and can be classified as MDR-TB. Thirty-three percent (13/39) of the persistant tuberculosis cases (5/18 treatment failure; 5/14 relapse; 3/7 defaulter) had mutations accounting for drug resistance. A total of 27 different spoligotypes were identified from 49/50 samples. Twenty-nine (59%) of the isolates were grouped into one of seven clusters, while 20 (41%) showed unique patterns. One patient was infected with M. bovis. CONCLUSION: This is the first molecular approach to characterize clinical isolates of M. tuberculosis from Sudan. The results show that drug resistance is indeed a serious problem and it may compliment the efforts of the National Tuberculosis Programme to improve strategies to control this disease.

Synthesis, spectroscopic characterization, stability assessment and DNA-binding of new 2,6-piperidinedione derivatives.

This work reports on structural characterization of new antineoplaston (ANP) representatives, namely 3-(benzoylamino)-2,6-piperidinedione (BPD), 3-(4-methoxybenzoylamino)-2,6-piperidinedione (MPD) and 3-(p-nitrobenzoylamino)-2,6-piperidinedione (NPD). These compounds were prepared by reacting N-(4-substituted benzoyl)-glutamines with N-hydroxysuccinimide to afford the corresponding esters, which were heated to produce the corresponding 2,6-piperidinedione (PD) compounds. Non-destructive analytical procedures such as 1H NMR and NIR analyses confirmed the postulated chemical structures of these PD compounds. HPLC chromatograms at an ambient temperature or from solutions preheated at 30, 40 or 60 degrees C displayed only a single peak for each compound. Combination of heat with pH modification had virtually no effect on the obtained peaks, thus attesting to the stability and purity of these compounds. MS analysis displayed molecular mass ions indicative of BPD, MPD and NPD at m/z 233.4, 263.2 and 278.3, respectively. The fragmentation patterns using MS/MS analyses conformed to the structural and molecular formulae of the prepared compounds. Furthermore, preliminary biological assessments showed the capacity of these compounds to bind to the DNA. NPD, but not BMP or MPD, had a superior affinity to the DNA than the prototype ANP-A10.

Determination of linezolid in human plasma by LC-MS-MS.

A rapid, sensitive and selective LC-atmospheric pressure-chemical ionization-MS-MS method for the determination of the new antimicrobial agent, linezolid, in human plasma using selected reaction monitoring (SRM) was developed. Linezolid and the internal standard were extracted from the biological samples by solid phase extraction (SPE) and analyzed on a reversed-phase Shim Pack CLC-CN, C18 column with the mobile phase of acetonitrile and 20 mM ammonium acetate solution (4 + 1 v/v). Detection was accomplished using an LCQ mass spectrometer (Finnigan), which was programmed in positive MS-MS mode to permit measurement of the fragment ions of linezolid and internal standard at m/z 296.2 and 223.2, respectively. The assay run-time was less than 3.5 min. Quantitative analysis was based on peak area ratio of linezolid to the internal standard. Calibration plots were established over the concentration range of 0.1-20 micrograms ml-1 of linezolid with the lowest detection limit of 0.05 microgram ml-1 using 10 microliters sample volume. The SPE technique quantitatively recovered linezolid and the internal standard from the plasma samples at a percentage range of 89.1-93.7%. Determination of control samples of linezolid in plasma validated the LC-MS-MS-SRM method. Intra-assay and inter-assay precision were in the range of 5.1-11.4% relative standard deviation, whereas the intra- and inter-accuracy were in the range of 97.5-114.0% of the nominal concentrations of linezolid added. The data confirmed that the plasma samples of linezolid were stable at room temperature and when stored at -20 degrees C for at least 10 d. The developed LC-MS-MS-SRM method is recommended for the determination of linezolid in human plasma.

Determination of diclofenac sodium, flufenamic acid, indomethacin and ketoprofen by LC-APCI-MS.

A sensitive, selective and accurate high-performance liquid chromatography-mass spectrometry (LC-MS) assay for the determination of selected non-steroidal anti-inflammatory drugs (NSAIDs), namely diclofenac sodium (DIC), flufenamic acid (FLU), indomethacin (IND) and ketoprofen (KET), either individually or in mixtures, was developed. The examined drugs were injected onto Shim-pack GLC-CN column and were eluted with a mobile phase consisting of acetonitrile and 20 mM ammonium acetate solution (5:1 v/v)/pH 7.4 at a flow rate l ml min(-1). The mass spectrometer, operated in the single ion monitoring mode, was programmed to admit the negative ions [M-H] at m/z 295.9 (DIC), 280.1 (FLU), 355.8 (IND) and 252.9 (KET), respectively. The calibration curves were linear (r > or = 0.9993) over the concentration range 50-300 ng ml(-1) (FLU, DIC) and 100-500 ng ml(-1) (KET, IND) with detection limits of 0.5-4.0 ng. The mean predicted concentrations for the analytes were in the range -5.9 and 5.2% of the nominal concentrations. Within-day and between-day precision were in the range of 0.8-9.1% of the R.S.D. Mean recovery percentages of the individual compounds from laboratory-made mixtures and pharmaceutical formulations were (99.5-101.5%) and (100.6-102.2%), respectively.

Nocardia africana sp. nov., a new pathogen isolated from patients with pulmonary infections.

Eight actinomycete strains, isolated from 8 out of 400 sputum samples examined, taken from patients with pulmonary diseases at the Chest Unit of Khartoum Teaching Hospital in the Sudan, were provisionally assigned to the genus Nocardia according to morphological criteria. These isolates were studied further in order to establish their taxonomic status. They were found to have morphological and chemical properties typical of nocardiae and formed a monophyletic clade in the 16S ribosomal DNA tree together with Nocardia vaccinii. The strains showed a unique pattern of phenotypic properties that distinguished them from representatives of recognized Nocardia species, including Nocardia vaccinii. The strains were considered to merit species status and were designated Nocardia africana sp. nov. The findings of the present study are consistent with the view that pulmonary nocardiosis may occur in a substantial proportion of patients who exhibit chronic lung diseases in African countries. It is important, therefore, that clinicians in such countries consider this condition, especially when patients with respiratory infections fail to respond to antitubercular therapy.

Comparative LC-MS and HPLC analyses of selected antiepileptics and beta-blocking drugs.

A highly sensitive and specific assay procedure based on the combination of liquid chromatography and mass spectrometry (LC-MS) has been developed for the quantitative analysis of selected antiepileptics (carbamazepine and phenytoin) and beta-blocking drugs (acebutolol, atenolol, pindolol and propranolol) using APCI as an ionization process. The measured concentration range was 100-300 ng ml-1 for all drugs except phenytoin (0.5-1.5 micrograms ml-1). Analysis was based on direct injection of methanolic solutions of drugs into the mass spectrometer with the subsequent elution with a mobile phase consisting of methanol and 1% acetic acid solution (4:1) at a flow rate 1 ml min-1. The mass spectrometer was programmed to permit detection and determination of either fragment or molecular ions of carbamazepine, phenytoin, acebutolol, atenolol, pindolol and propranolol at m/e 194.3, 252.9, 337.2, 267.1, 249.1 and 260.1, respectively. The recorded chromatograms exhibited well-resolved peaks at retention times < 1 min. The peak area was correlated linearly to the drug concentration. Intraday precision gave relative standard deviations in the range 1.75-4.02%. Compared to HPLC, the described LC-MS was faster, more sensitive and specific. Unlike HPLC, LC-MS could be applied to analyze incompletely resolved mixtures. The absolute detection limits for LC-MS and HPLC were 0.2-0.5 and 10-25 ng, respectively. Recovery studies of the investigated compounds in pharmaceutical products using LC-MS and HPLC gave mean percentages of 97.5-102.0 and 98.4-103.3, respectively. Statistical analysis of the data using t- and F-tests showed insignificant differences between both methods for the analysis of carbamazepine, phenytoin, acebutolol and atenolol in pharmaceutical formulations. However, LC-MS gave more accurate results than HPLC for determination of pindolol in tablets. Propranolol could only be determined in tablets using LC-MS.

High-performance liquid chromatography-mass spectrometric analysis of furosemide in plasma and its use in pharmacokinetic studies.

This study presents a rapid, specific and sensitive high-performance liquid chromatography-mass spectrometric (LC-MS) assay for the determination of furosemide in human plasma using diclofenac as an internal standard (IS). Both compounds were extracted from human plasma with ethyl acetate at pH 1 and were chromatographed using Shim-Pack GLC-CN column and a mobile phase consisting of acetonitrile and 20 mM ammonium acetate buffer solution pH 7, 4:1 (v/v) at a flow rate 1 ml min(-1). Furosemide and IS were detected by mass spectrometer operated in the negative single ion monitoring mode using APCI as an ionization process at m/z 329.2 and 294.1, respectively. The assay linearity of furosemide was confirmed over the range 50-2,000 ng ml(-1). Detection limit for furosemide in plasma was 10 ng ml(-1). The selected concentration range corresponds well with the plasma concentrations of furosemide for pharmacokinetic study. Intraday and interday relative standard deviations were 1.3-4.7 and 2.7-11.5%, respectively. The extraction recovery percentages of furosemide and IS from plasma were in the range 89.3-97.1%. The developed LC-MS procedure was applied for the determination of the pharmacokinetic parameters of furosemide after an oral administration of tablet formulation (40 mg) to two healthy male volunteers. The calculated parameters were in good agreement with the reported values.

Total salivary gland proteins of female Culex pipiens and Aedes caspius (Diptera: Culicidae) and their fractionation during adult development and after blood sucking.

Salivary glands of Culex pipiens and Aedes caspius were analyzed to determine total protein content and its fractionation during adult female development and after blood sucking. In both species, the molecular weight of proteins ranged between 26.000 and 84.000 Daltons. These proteins were not identical in the two species. In Cx. pipiens, the total protein level increased during the first 3 days of adult development from 4.94 +/- 0.84 to 6.6 +/- 0.37 micrograms/gland. During this period, the salivary gland proteins were separated into 35, 34 and 37 fractions respectively. Cx. pipiens released in the human host 64% of the total proteins while taking a blood meal compared to unfed females. This decrease in protein level was proportional to protein fractions. Over the next 6 days, the protein level increased again to attain values comparable to those obtained prior to blood sucking. In Ae. caspius, the total protein level of the salivary glands did not change during the first 4 days of adult development (range between 3.13 +/- 0.27 and 3.91 +/- 0.36 micrograms gland), but on the fifth day, 2-fold increase was observed. The total salivary gland protein increased during the next 3 days after blood sucking to reach 15.5 +/- 0.98 micrograms/gland. During this period, a tremendous change in protein patterns was observed. After oviposition, on the fourth day, a significant reduction in the total protein level was observed (4.13 +/- 0.56 micrograms/gland), but over the next 3 days the level increased again (range between 4.13 +/- 0.66 and 7.13 +/- 0.66 micrograms/gland).