Fatal Nocardia paucivorans Lower Respiratory Tract Infection in a Tuberculosis Suspect Who Worked as Gold Miner.

Nocardiosis is an opportunistic infection that affects both immunocompromised as well as immunocompetent patients. The main infections occur as soft tissue and lung infections although they might disseminate to various organs. This is a case study aimed to reflect the severity of the disease and the patient's risk factors associated with the infection. A sputum sample was collected from tuberculosis (TB) suspects for culture. Nocardia-like colonies were isolated, purified, and sent to BGI Company (Hongkong, China). Standard forward sequencing of 16S rRNA was done by ABI Genetic Analyzer (Applied Biosystems). Sequence alignment and nucleotide basic local alignment search tool (BLAST) were done using National Center for Bioinformatics (NCBI) Nucleotide BLAST. Biochemical identification to the colonies was done using an automation system (BD Phoenix™) to confirm the identification. Nocardia paucivorans was identified from the TB suspect. Risk factors were identified as extensive contact to dust, absence of primary care units with complete facilities, and old age. Since the infection of the lungs caused by Nocardia might be similar to pulmonary TB, this case report highlights the importance of accurate diagnosis and identification procedures to differentiate between the two.

Molecular Determination of Tumor Necrosis Factor-alpha, Interleukin-8, Interleukin-10, and C-X-C Chemokine Receptor-2 Genetic Variations and their Association with Disease Susceptibility and Mortality in COVID-19 Patients.

Background: Altered cytokine levels have been associated with poor outcomes among COVID-19 patients. TNF-α, IL-8 and IL-10 are key cytokines in COVID-19 pathogenesis, and CXCR-2 is a major chemokine receptor involved in inflammatory response. Polymorphisms in the genes of these proteins are proposed to influence disease outcomes. In this study, we aimed to find out the association of genetic polymorphisms in TNF-α, IL-8, IL-10 and CXCR-2 genes with susceptibility to and mortality of COVID-19. Methods: The present case-control study was conducted on 230 subjects, among whom 115 were clinically diagnosed and RT-PCR-confirmed COVID-19 patients and 115 healthy control subjects. The polymorphisms in TNFα -308 G>A (rs1800629), IL-8 -251T>A (rs4073), CXCR2 +785 C>T (rs2230054) genes were detected by ARMS -PCR assay whereas for IL-10 (-1082 G>A), rs1800896 G>A allele-specific PCR assay was used and their association with COVID-19 susceptibility and mortality was estimated by multivariate analysis. The results were analyzed for risk of infection and mortality through different inheritance models. Results: Frequencies of TNF-α rs1800629 GA, AA, IL-8 rs4073 TA, AA, IL-10 (-1082 G>A), rs1800896 GA and GG, and CXCR2 rs2230054 CT genotypes were significantly higher in COVID-19 patients compared to the control group (p < 0.05). Furthermore, COVID-19 patients had a higher frequency of the polymorphic A allele of TNF-α, the A allele of IL-8, the G allele of IL-10, and the T allele of CXCR2. The risk of susceptibility to COVID-19 was significantly associated with TNF-α rs1800629 GA, GA+AA genotypes and the A allele, IL-8 rs4073 TA, AA genotypes and A allele, IL-10 rs1800872 GA and CC genotypes and C allele, and CXCR2 rs2230054 CT and CT+CC genotypes. TNF-α-GA and AA genotypes and A allele, IL-8 TA and AA genotypes and A allele and CXCR-2 CC and CT genotypes have significant associations with mortality risk in COVID-19 patients, while GA and GG genotypes of the IL-10 are shown to confer significant protection against mortality from COVID-19. Conclusion: The findings of this study provide important insights into the COVID-19 disease and susceptibility risk. The polymorphisms in TNFα -308 G>A (rs1800629), IL-8 -251T>A (rs4073), IL-10 (-1082 G>A), rs1800896 and CXCR2 +785 C>T (rs2230054) are associated with the risk of susceptibility to COVID-19 and with mortality in COVID-19 patients. Further studies with larger sample sizes are necessary to confirm our findings.

COVID-19: Establishing, implementing and assessment safety strategy, descriptive interventional study.

The increase in severe acute respiratory syndrome SARS-CoV-2 has invariably affected medical professionals in their training, academic and professional development. The present study was an interventional descriptive study aimed at reducing the risk of exposure to COVID-19 and enabling physical attendance to the practical session for applied medical students by establishing and implementing a safety strategy. The adopted safety strategy has eight conditions and 50 requirements. Compliance with the safety strategy along with the serological diagnosis of COVID-19 was used as a key performance indicator for assessing the efficiency of the safety strategy. A total of 197 students were enrolled at the beginning of the study. The overall results showed that 78.1% of the respondents strictly followed the protocol, 14.5% of the individuals partially responded to the protocol and 7.4% of the individuals did not respond to the protocol. Twenty-two (12.6%) out of the 175 participants who completed the study had positive COVID-19 during the study period, whereas the remaining 153 participants (87.4%) appeared to be healthy. The serological results showed that 68 (38.9%) and 66 (37.7%) individuals of the study population had positive IgM+IgA and IgG of COVID-19, respectively; the majority of the participants who developed antibodies did not show symptoms and appeared to be healthy during the study. The physical distancing condition was the only condition that displayed a significant relationship with seropositive IgM+IgA. The compiling of standardized protocols along with serological diagnoses can be an effective tool in measuring the effectiveness of safety protocol and reducing the risk of exposure to COVID-19.

Omicron SARS-CoV-2 Variants in an In Silico Genomic Comparison Study with the Original Wuhan Strain and WHO-Recognized Variants of Concern.

This study aimed to determine the genetic alterations in the Omicron variants compared to other variants of concern (VOCs) to trace the evolutionary genetics of the SARS-CoV-2 variants responsible for the multiple COVID-19 waves globally. The present study is an in silico analysis determining the evolution of selected 11 VOCs compared to the original Wuhan strain. The variants included six Omicrons and one variant of Alpha, Beta, Delta, Gamma, and Mu. The pairwise alignment with the local alignment search tool of NCBI Nucleotide-BLAST and NCBI Protein-BLAST were used to determine the nucleotide base changes and corresponding amino acid changes in proteins, respectively. The genomic analysis revealed 210 nucleotide changes; most of these changes (127/210, 60.5%) were non-synonymous mutations that occurred mainly in the S gene (52/127, 40.1%). The remaining 10.5% (22/210) and 1.9% (4/210) of the mutations were frameshift deletions and frameshift insertions, respectively. The frameshift insertion (Ins22194T T22195G) led to frameshift deletion (Δ211N). Only four mutations (C241T, C3037T, C14408T, and A23403G) were shared among all the VOCs. The nucleotide changes among Omicron variants resulted in 61 amino acid changes, while the nucleotide changes in other VOCs showed 11 amino acid changes. The present study showed that most mutations (38/61, 62.3%) among Omicron variants occurred in the S gene; and 34.2% of them (13/38) occurred in the receptor-binding domain. The present study confirmed that most of mutations developed by Omicron variants occurred in the vaccine target gene (S gene).

Clinical Implications of Glyoxalase1 Gene Polymorphism and Elevated Levels of the Reactive Metabolite Methylglyoxal in the Susceptibility of Type 2 Diabetes Mellitus in the Patients from Asir and Tabuk Regions of Saudi Arabia.

Diabetes mellitus constitutes a big challenge to the global health care system due to its socioeconomic impacts and very serious complications. The incidence and the prevalence rate are increased in the Gulf region including the KSA. Type 2 diabetes mellitus (T2DM) is caused by diverse risk factors including obesity, unhealthy dietary habits, physical inactivity, smoking and genetic factors. The molecular genetic studies have helped in the detection of many single nucleotide polymorphisms (SNP) with different diseases including cancers, cardiovascular diseases and T2DM. The glyoxalase 1 (GLO1) is a detoxifying enzyme and catalyzes the elimination of the cytotoxic product methylglyoxal (MG) by converting it to D-lactate, which is not toxic to tissues. MG accumulation is associated with the pathogenesis of different diseases including T2DM. In this study, we have investigated the association of the glyoxalase 1 SNPs (rs2736654) rs4746 C>A and rs1130534 T>A with T2DM using the amplification refractory mutation system PCR. We also measured the concentration of MG by ELISA in T2DM patients and matched heathy controls. Results show that the CA genotype of the GLO rs4647 A>C was associated with T2DM with OR = 2.57, p-value 0.0008 and the C allele was also associated with increased risk to T2DM with OR = 2.24, p-value = 0.0001. It was also observed that AT genotype of the rs1130534 was associated with decreased susceptibility to T2DM with OR = 0.3, p-value = 0.02. The A allele of rs1130534 was also associated with reduced risk to T2DM with PR = 0.27 = 0.006. In addition, our ELISA results demonstrate significantly increased MG concentrations in serum of the T2DM patients. We conclude that the GLO1 SNP may be associated with decreased enzyme activity and a resultant susceptibility to T2DM. Further well-designed studies in different and large patient populations are recommended to verify these findings.

In-silico genomic landscape characterization and evolution of SARS-CoV-2 variants isolated in India shows significant drift with high frequency of mutations.

In-silico studies on SARS-CoV-2 genome are considered important to identify the significant pattern of variations and its possible effects on the structural and functional characteristics of the virus. The current study determined such genetic variations and their possible impact among SARS-CoV-2 variants isolated in India. A total of 546 SARS-CoV-2 genomic sequences (India) were retrieved from the gene bank (NCBI) and subjected to alignment against the Wuhan variant (NC_045512.2), the corresponding amino acid changes were analyzed using NCBI Protein-BLAST. These 546 variants revealed 841 mutations; most of these were non-synonymous 464/841 (55.1%), there was no identical variant compared to the original strain. All genes; coding and non-coding showed nucleotide changes, most of the structural genes showed frequent nonsynonymous mutations. The most affected genes were ORF1a/b followed by the S gene which showed 515/841 (61.2%) and 120/841 (14.3%) mutations, respectively. The most frequent non-synonymous mutation 486/546 (89.01%) occurred in the S gene (structural gene) at position 23,403 where A changed to G leading to the replacement of aspartic acid by glycine in position (D614G). Interestingly, four variants also showed deletion. The variants MT800923 and MT800925 showed 12 consecutive nucleotide deletion in position 21982-21993 resulting in 4 consecutive amino acid deletions that were leucine, glycine, valine, and tyrosine in positions 141, 142, 143, and 144 respectively. The present study exhibited a higher mutations rate per variant compared to other studies carried out in India.

Anticancer Activity of Extremely Effective Recombinant L-Asparaginase from Burkholderia pseudomallei.

L-asparaginase (E.C. 3.5.1.1) purified from bacterial cells is widely used in the food industry, as well as in the treatment of childhood acute lymphoblastic leukemia. In the present study, the Burkholderia pseudomallei L-asparaginase gene was cloned into the pGEX-2T DNA plasmid, expressed in E. coli BL21 (DE3) pLysS, and purified to homogeneity using Glutathione Sepharose chromatography with 7.26 purification fold and 16.01% recovery. The purified enzyme exhibited a molecular weight of ~33.6 kDa with SDS-PAGE and showed maximal activity at 50°C and pH 8.0. It retained 95.1, 89.6%, and 70.2% initial activity after 60 min at 30°C, 40°C, and 50°C, respectively. The enzyme reserved its activity at 30°C and 37°C up to 24 h. The enzyme had optimum pH of 8 and reserved 50% activity up to 24 h. The recombinant enzyme showed the highest substrate specificity towards L-asparaginase substrate, while no detectable specificity was observed for L-glutamine, urea, and acrylamide at 10 mM concentration. THP-1, a human leukemia cell line, displayed significant morphological alterations after being treated with recombinant L-asparaginase and the IC50 of the purified enzyme was recorded as 0.8 IU. Furthermore, the purified recombinant L-asparaginase improved cytotoxicity in liver cancer HepG2 and breast cancer MCF-7 cell lines, with IC50 values of 1.53 and 18 IU, respectively.

Characterization of altered genomic landscape of SARS-CoV-2 variants isolated in Saudi Arabia in a comparative in silico study.

SARS-CoV-2 has become one of the unprecedented global health challenge for human population. Genomic signature studies of SARS-CoV-2 reveals relation between geographical location of the isolates and genetic diversity. The present work is an in silico, cross sectional study aimed to determine the genetic heterogeneity of SARS-CoV-2 variants isolated in Saudi Arabia compared to the first isolated strain NC_045512 (reference sequence). Each sequence was aligned against the reference sequence using local alignment search tool (NCBI) Nucleotide-BLAST. A total of 58 SARS-CoV-2 genomes were isolated in KSA and retrieved from NCBI. Our study shows that KSA variants demonstrated homology ranging between 99.96 and 99.98 % compared to the reference strain. There are 89 nucleotide changes that have been identified among the KSA variants; the most common nucleotide change was C: T accounting for 50.6% (45/89). These nucleotides changes resulted in 53.9% (48/89) missense mutations and 42.7% (38/89) silent mutations; while the majority of mutations- 48.3% (43/89) occurred in ORF1ab gene. All structural genes displayed mutations; N gene harbored 16.9% (15/89) mutations, S gene displayed 15.7% (14/89) mutations, M gene exhibited 2.2% (2/89) mutations and E gene showed only 1 mutation which was silent. The most frequently changed nucleotide was C3037T (silent mutation) and A23403G (D614G), each of which occurred in 57 variants out of 58 followed by C14408T (P4715L) and C241T (5'UTR) which were found in 56 and 55 variants respectively. The Phylogenetic trees showed that SARS-CoV-2 variants isolated in Saudi Arabia clustered together closely.

Genetic variations among SARS-CoV-2 strains isolated in China.

The rapid spread of COVID-19, which has led to a global pandemic, has placed public health systems under severe pressure. Identifying variations in SARS-CoV-2 strains from different regions is a key factor for understanding the pathogenic mechanisms, aid in diagnosis, prevention and therapy of this disease. The present study is an analytical descriptive study aimed to determine genetic variations among SARS-CoV-2 strains isolated in China. Sixty six complete genome sequences of the virus were retrieved from NCBI, the sequence of original Wuhan strain accession number NC 045512 was used as the reference sequence. Each genome sequence was blasted against the original Wuhan strain; the analysis was done using NCBI Nucleo-blast. The collected sequences showed 10 different variants. One hundred and thirty four mutations were identified among the variants of SARS-CoV-2 in this study; most of them 52.2% (70/134) were missense point mutation, majority of the mutations 65.7% (88/134) occurred in the open reading frame a/b (ORFab), few mutations occurred in the structural viral genome, each of spike (S) gene and nucleocapsid (N) gene showed 4 mutations; 2 silent point mutations and 2 missense point mutations occurred in each gene whereas membrane (M) gene showed silent point mutation and no mutation observed in the envelope E gene. The remarkable observation in this study showed by Yunnan variant accession number MT226610 which exhibited high incidence of mutations, it displayed 28 different point mutations; only 3(10.7%) of them were silent mutations while the rest were missense mutations. Our analysis showed several mutations including spike S gene and membrane M gene which may be responsible for a change in the structures of target proteins.

Multiplex PCR is a Rapid, Simple and Cheap Method for Direct Diagnosis of M. tuberculosis from Sputum Samples.

INTRODUCTION: Rapid diagnosis of M. tuberculosis directly from sputum samples is a challenging process. This study aimed to design and evaluate a multiplex-PCR method for direct diagnosis of M. tuberculosis from sputum specimens. MATERIALS AND METHODS: 46 suspected tuberculosis patients and 25 apparently healthy individuals were enrolled in the study. Sputa were collected from the study population and processed by cold ZN stain. DNA was extracted from each sample and processed by Multiplex PCR and Genotype Mycobacteria CM. RESULTS: Out of the 46 Tuberculosis suspected patients, 22 (47.8%) revealed positive Acid fast ba- cilli (AFB), while 19 (41.3%) showed positive by both multiplex PCR and Genotype Mycobacte- ria CM. The overall sensitivity of multiplex PCR and smear microscopy were 100% while the specificity were 100, and 86.3%, respectively. CONCLUSION: Multiplex PCR method using two different sets of primers in combination with other diagnostic tools such as X-Rays and smear Microscopy are cheap, rapid and reliable methods for the diagnosis of M. tuberculosis from clinical samples and are able to identify most of the smear positive cases with valuable accuracy.

Emergence of Heteroresistance Mycobacterium Tuberculosis in Saudi Arabia.

PURPOSE: Heteroresistant Mycobacterium tuberculosis (MTB) is defined as a group of drug-susceptible and resistant bacteria in a single clinical specimen from tuberculosis (TB) patients. Heteroresistance of MTB is considered a preliminary stage to full resistance. The present study aimed to determine the heteroresistance in Mycobacterium tuberculosis in Tabuk province, in the north of the Kingdom of Saudi Arabia. METHOD: GenoType MTBDRplus assay was used to determine mutations associated with isoniazid and rifampicin resistance. RESULTS: A total number of 46 confirmed M. tuberculosis positive sputum samples were scanned for heteroresistance. The present study revealed 3 (6.5%) heteroresistant mutations to either rpoB gene alone, 2 (4.4%) to rpoB and 1 (2.2%) to inhA genes. CONCLUSION: The detection of heteroresistant mutations could guide the initiation of an appropriate regimen of treatment.

Detection of multidrug resistant Mycobacterium tuberculosis in Tabuk, Saudi Arabia, using Genotype MTBDRplus.

BACKGROUND: The global increase in the rates of multidrug-resistant (MDR) tuberculosis (TB) has made the timely identification of resistant Mycobacterium tuberculosis complex strains an important emergence to achieve effective disease management and to prevent their spread in the community. The present study aimed to determine the MDR-TB in Tabuk province, north of the Kingdom of Saudi Arabia. METHODS: The GenoType MTBDRplus assay was used to determine the mutations associated with isoniazid (INH) and rifampicin (RIF) resistances. A total number of 61 confirmed M. tuberculosis positive-sputum samples were scanned for the mutation in the rpoB, inhA, and katG genes. RESULTS: The present study revealed that 67.2% of the samples were susceptible, 29.5% were monoresistant, and 3.3% were MDR. CONCLUSIONS: The monoresistant showed 26.2% for INH and 3.3% for RIF. The early detection of MDR could guide the starting of appropriate regimen of treatment.

Smear microscopy as a diagnostic tool of tuberculosis: Review of smear negative cases, frequency, risk factors, and prevention criteria.

Tuberculosis is one of the global health problems, the estimated deaths due to TB was around 2 million in the year 2013. Failure in early diagnosis and providing suitable treatment leads to increase the prognosis of the disease. Smear microscopy is used in many countries as a primary diagnosis of TB especially in the district poor facility laboratories, where smear negative frequency is high. This review aimed to reflects the importance of smear negative tuberculosis as a source of infection and poor prognosis of TB treatment and prevention. In addition to, discuss the possible causes and suggests solutions to improve the yields of smear microscopy.

Prevalence and antimicrobial resistance pattern of bacterial strains isolated from patients with urinary tract infection in Messalata Central Hospital, Libya.

OBJECTIVES: To investigate the prevalence of urinary tract infection among patients at Messalata Central Hospital, Libya, to identify the causative bacteria, and to explore their resistance pattern to antimicrobials. METHODS: A total number of 1153 urine samples were collected from patients, who attended daily to Messalata Central Hospital, Libya, in a study extended for one year. Antimicrobial susceptibility testing and isolates typing were done using Phoenix BD (BD diagnostic). Resistance was confirmed manually using agar disk diffusion method. RESULTS: Of the 1153 urine samples tested, 160 (13.9%) samples were positive, from which 17 different, solely Gram negative, uropathogens were identified. Escherichia coli were the most prevalent (55.6%) bacteria, followed by Klebsiella pneumoniae subspecies pneumoniae (16.3%), Proteus mirabilis (6.3%), Pseudomonas aeruginosa (5.6%), Enterobacter cloacae and Klebsiella oxytoca (2.5%, each), Citrobacter koseri and Providencia rettgeri (1.9%, each), Acinetobacter baumannii, Enterobacter aerogenes and Proteus vulgaris (1.3%, each), and Aeromonas caviae, Citrobacter freundii, Cronobacter sakazakii, Enterobacter amnigenus biogroup 2, Pseudomonas putida and Serratia marcescens (0.6%, each). The isolated uropathogens showed increased levels of resistance ranged from 10.5% to 64.5%, with an overall resistance of 28.9%. Amikacin was the most effective antimicrobial followed by Imipenem and Meropenem (0%, 0.6% and 2.5% resistance, respectively); while, Cephalothin and Ampicillin were the least (80.6% and 90.0% resistance, respectively) effective. CONCLUSIONS: The obtained results emphasized the emergence of highly resistant bacteria to most of tested antimicrobials and raise the alarm for physicians to change their treatment pattern depending on antimicrobial susceptibility results.

Comparison of Xpert MTB/RIF with ProbeTec ET DTB and COBAS TaqMan MTB for direct detection of M. tuberculosis complex in respiratory specimens.

BACKGROUND: Nucleic acid amplification assays allow for the rapid and accurate detection of Mycobacterium tuberculosis (MTB) directly in clinical specimens thereby facilitating diagnosis of tuberculosis (TB). With the fully automated Xpert MTB/RIF system (Cepheid) an innovative solution of TB diagnostics has been launched. We performed a direct head-to-head comparison of Xpert MTB/RIF with two widely used commercial assays, ProbeTec ET DTB (DTB) (Becton-Dickinson) and COBAS TaqMan MTB (CTM-MTB) (Roche). METHODS: 121 pre-characterized respiratory specimens (68 culture-positive for MTB complex, 24 culture-positive for non-tuberculous mycobacteria and 29 culture-negative) taken from our frozen specimen bank were tested for the presence of MTB complex by the three assays. RESULTS: Among culture-positive samples (n = 68), overall sensitivity for detection of MTB complex was 74.6%, 73.8%, and 79.1% for Xpert MTB/RIF, CTM-MTB, and DTB, respectively. Within the subgroup of smear-negative TB samples (n = 51) sensitivity was 68% for Xpert MTB/RIF and CTM-MTB and 72% for DTB. Among smear-positive TB samples (n = 17), all (100%) were detected by DTB and 94.1% and 93.3% by Xpert MTB/RIF and CTM-MTB, respectively. Specificity was best for CTM-MTB (100%) and lowest for Xpert MTB/RIF (96.2%) due to misidentification of two NTM samples as MTB complex. CTM-MTB yielded the highest rate of invalid results (4.1%) (0.8% by Xpert MTB/RIF and DTB, respectively). CONCLUSIONS: The direct comparison of Xpert MTB/RIF with CTM-MTB and DTB yielded similar overall performance data. Whereas DTB was slightly superior to Xpert MTB/RIF in terms of sensitivity, at least in the sample collection tested here, CTM-MTB performed best in terms of specificity.

Multi-centre evaluation of the speed-oligo Mycobacteria assay for differentiation of Mycobacterium spp. in clinical isolates.

BACKGROUND: A new DNA line probe assay (Speed-oligo Mycobacteria, Vircell) has been launched for rapid differentiation of Mycobacterium spp. from cultures. Compared to other line-probe assays, Speed-oligo Mycobacteria covers a relatively limited spectrum of species but uses a simpler and faster dip-stick technique. The present multi-centre, multi-country study aimed at evaluating the utility and usability of Speed-oligo Mycobacteria in routine mycobacteriology diagnostics. Results from Speed-oligo Myobacteria were compared to those from Genotype CM (HAIN lifescience, Nehren, Germany), another line-probe assay. METHODS: Speed-oligo Mycobacteria assay was performed in three main steps: 1) DNA extraction from cultured material 2) PCR amplification of the target gene and an internal control and 3) hybridization of the PCR products to specific probes by means of a dip-stick. RESULTS: Two hundred forty-two clinical isolates were recovered from consecutive positive mycobacterial cultures at two German (IML Gauting, Bioscientia Ingelheim), one Czech (KLINLAB Prague), and at a Sudanese (Khartoum) laboratory. All Mycobacterium species covered by the assay were reliably recognized. The rate of false positive results was 1.2% and concerned only the species M. marinum and M. peregrinum. The identification rate, i.e. the proportion of isolates which was correctly differentiated to the level of species or complex by the assay, differed significantly among laboratories being 94.9%, 90.7%, and 75.0% at the study sites IML Gauting, KLINLAB Prague and Bioscientia Ingelheim, respectively. This difference was caused by different spectra of NTM species encountered by the laboratory centres in daily routine diagnostics. CONCLUSIONS: Speed-oligo Mycobacteria assay was proved a rapid and easy-to-perform alternative to conventional line-probe assays. The assay showed excellent sensitivity with regard to identification of genus Mycobacterium and species/complexes covered by the test. However, due to its relatively limited spectrum of taxa, a varying proportion of NTM may not be identified by the assay in daily diagnostics demanding further analyses. The only significant shortcoming in terms of specificity was the misidentification of the clinically relevant species M. marinum.