5-Substituted 2-benzylidene-1-tetralone analogues as A1 and/or A2A antagonists for the potential treatment of neurological conditions.

Adenosine A1 and A2A receptors are attracting great interest as drug targets for their role in cognitive and motor deficits, respectively. Antagonism of both these adenosine receptors may offer therapeutic benefits in complex neurological diseases, such as Alzheimer's and Parkinson's disease. The aim of this study was to explore the affinity and selectivity of 2-benzylidene-1-tetralone derivatives as adenosine A1 and A2A receptor antagonists. Several 5-hydroxy substituted 2-benzylidene-1-tetralone analogues with substituents on ring B were synthesized and assessed as antagonists of the adenosine A1 and A2A receptors via radioligand binding assays. The results indicated that hydroxy substitution in the meta and para position of phenyl ring B, displayed the highest selectivity and affinity for the adenosine A1 receptor with Ki values in the low micromolar range. Replacement of ring B with a 2-amino-pyrimidine moiety led to compound 12 with an increase of affinity and selectivity for the adenosine A2A receptor. These substitution patterns led to enhanced adenosine A1 and A2A receptor binding affinity. The para-substituted 5-hydroxy analogue 3 behaved as an adenosine A1 receptor antagonists in a GTP shift assay performed with rat whole brain membranes expressing adenosine A1 receptors. In conclusion, compounds 3 and 12, showed the best adenosine A1 and A2A receptor affinity respectively, and therefore represent novel adenosine receptor antagonists that may have potential with further structural modifications as drug candidates for neurological disorders.

Qualitative and quantitative detection of SARS-CoV-2 RNA in untreated wastewater in Western Cape Province, South Africa.

Recent studies have shown that the detection of SARS-CoV-2 genetic material in wastewater may provide the basis for a surveillance system to track the environmental dissemination of this virus in communities. An effective wastewater-based epidemiology (WBE) system may prove critical in South Africa (SA), where health systems infrastructure, testing capacity, personal protective equipment and human resource capacity are constrained. In this proof-of-concept study, we investigated the potential of SARS-CoV-2 RNA surveillance in untreated wastewater as the basis for a system to monitor COVID-19 prevalence in the population, an early warning system for increased transmission, and a monitoring system to assess the effectiveness of interventions. The laboratory confirmed the presence (qualitative analysis) and determined the RNA copy number of SARS-CoV-2 viral RNA by reverse transcription polymerase chain reaction (quantitative) analysis from 24-hour composite samples collected on 18 June 2020 from five wastewater treatment plants in Western Cape Province, SA. The study has shown that a WBE system for monitoring the status and trends of COVID-19 mass infection in SA is viable, and its development and implementation may facilitate the rapid identification of hotspots for evidence-informed interventions.

Selected C8 two-chain linkers enhance the adenosine A1/A2A receptor affinity and selectivity of caffeine.

Recent research exploring C8 substitution on the caffeine core identified 8-(2-phenylethyl)-1,3,7-trimethylxanthine as a non-selective adenosine receptor antagonist. To elaborate further, we included various C8 two-chain-length linkers to enhance adenosine receptor affinity. The results indicated that the unsubstituted benzyloxy linker (1e A1Ki = 1.52 µM) displayed the highest affinity for the A1 adenosine receptor and the para-chloro-substituted phenoxymethyl (1d A2AKi = 1.33 µM) linker the best A2A adenosine receptor affinity. The position of the oxygen revealed that the phenoxymethyl linker favoured A1 adenosine receptor selectivity over the benzyloxy linker and, by introducing a para-chloro substituent, A2A adenosine receptor selectivity was obtained. Selected compounds (1c, 1e) behaved as A1 adenosine receptor antagonists in GTP shift assays and therefore represent selective and non-selective A1 and A2A adenosine receptor antagonists that may have potential for treating neurological disorders.

Investigation of isoniazid and ethionamide cross-resistance by whole genome sequencing and association with poor treatment outcomes of multidrug-resistant tuberculosis patients in South Africa.

OBJECTIVE/BACKGROUND: Ethionamide (ETH) and isoniazid (INH) are part of the backbone regimen used for the treatment of multidrug-resistant tuberculosis (MDR-TB). Both ETH and INH are structurally similar and are activated by ethA and katG gene products. Resistance to INH among MDR-TB patients may cause ETH to be ineffective, as both target nicotinamide adenine dinucleotide-dependent enoyl-acyl carrier protein reductase inhA protein and mutations within inhA gene may lead to their cross-resistance. Furthermore, ETH resistance is caused by mutations within ethA and ethR genes forming part of the ETH drug activation pathway. Nicotinamide adenine dinucleotide is coded by the ndh gene, and its overexpresion may lead cross-resistance between INH and ETH drugs. Phenotypic drug susceptibility testing of ETH is difficult and often unreliable. We used whole genome sequencing to compare inhA, inhA promoter, ethA, ethR ndh, and katG genetic regions in serial isolates (baseline and follow-up) with treatment outcomes. METHODS: MDR-TB strains were collected from 46 patients before and during second-line drug treatment in KwaZulu-Natal and Eastern Cape between 2005 and 2009. All patients had phenotypically determined MDR-TB at baseline and had treatment outcomes documented. Unfavorable treatment outcomes were defined as death, default, and failure, while favorable outcomes were cure and treatment completion. Each strain had baseline and at least one strain collected on follow-up. From each strain, DNA was extracted from colonies grown on Löwenstein-Jensen slants, and fragment and jumping paired-end Illumina DNA libraries were constructed and sequenced on the Illumina HiSeq 2000 (Broad Institute, Cambridge, MA, USA). Sequences were aligned to H37Rv genome and Pilon was run to generate a list of SNPs. In silico spoligotyping was performed to a database 43 unique spacer sequences. Cross-resistance was defined as the presence of both inhA and either ethA or ethR mutations in clinical isolates. RESULTS: A total of 92 sequences from 46 serial isolates of MDR-TB patients from KwaZulu-Natal (29 isolates) and Eastern Cape (17 isolates) were analyzed. Most patients (29/46; 63.0%) had unfavorable outcomes, 13 (28.3%) had favorable outcomes, while four (8.7%) had unknown outcomes. Phylogenetic reconstruction revealed that primary genotype differed by province. The Beijing genotype was predominant in Eastern Cape, while EuroAmerican lineage (S, T, LAM, X) was found in KwaZulu-Natal. Whole genome analysis revealed nonsynonymous insertions and deletions within katG, ethA, ethR, ndh, and inhA and its promoter region. Among patients with treatment outcome data, mutations were detected in 92.8% in katG, 50% in inhA, 53.6% in ethA, 2.4% in ethR, and 19% in ndh. The majority of mutations causing ETH (20/29; 68.9%) and INH (18/29; 62.1%) resistance occurred among patients with unfavorable outcomes. Both inhA and either ethA or ethR mutations were detected in 16/29 (55.2%) patients with unfavorable outcomes. Cross-resistance of both INH and ETH drugs was associated with unfavorable treatment outcomes (p=0.021) in 16/29 (55.2%) patients compared with favorable treatment outcomes in 2/13 (15.4%) patients. CONCLUSION: Baseline ETH molecular resistance before second-line treatment is a concern. Unfavorable treatment outcomes of patients with ethA, ethR, and inhA mutations highlight the importance of genotypic testing before initiation of treatment containing ETH. The clinical significance of whole genome analysis for early detection of mutations predictive of treatment failure needs further investigation.

Integration of TB-HIV services at an ANC facility in Frances Baard District, Northern Cape, South Africa.

BACKGROUND: Integrated tuberculosis-human immunodeficiency virus (TB-HIV) service delivery as part of maternal health services, including antenatal care (ANC), is widely recommended. This study assessed the implementation of collaborative TB-HIV service delivery at a hospital-based ANC service unit. METHODS: A record review of a random sample of 308 pregnant women attending the ANC service between April 2011 and February 2012 was conducted. Data were extracted from registers and patient case notes. Outcomes included the proportion of women who underwent HIV counselling and testing (HCT), CD4 count testing, antiretroviral treatment (ART), cotrimoxazole preventive treatment (CPT), TB screening and isoniazid preventive treatment (IPT). Analysis measured variations in patient characteristics associated with service delivery. RESULTS: All women underwent HCT; 80% of those who tested HIV-positive were screened for TB. Most (85.9%) of the HIV-positive women received a CD4 count. However, only 12.9% of eligible women received ART prophylaxis onsite, only 35.7% were referred for initiation of ART, only 42.3% commenced IPT and none received CPT or further investigations for TB. HIV-negative women had 2.6 higher odds (95%CI 1.3-5.3) of receiving TB screening than their HIV-positive counterparts. CONCLUSIONS: Although the identification of HIV-positive women and TB suspects was adequate, implementation of other TB-HIV collaborative activities was sub-optimal.

Contexte : Une offre de services intégrée de tuberculose et du virus de l'immunodéficience humaine (TB-VIH)­en tant qu'élément des services de santé maternelle, notamment des consultations prénatales (ANC)­est largement recommandée. Cette étude a évalué la mise en œuvre d'une offre de services intégrée TB-VIH dans une unité hospitalière de CPN.Méthodes : Les dossiers d'un échantillon aléatoire de 308 femmes enceintes qui ont fréquenté le service ANC entre avril 2011 et février 2012 ont été revus. Les données ont été extraites à partir des registres ainsi que des dossiers des patients. Les résultats attendus comprenaient la proportion de femmes bénéficiant d'un conseil et test VIH (HCT), d'un comptage des CD4, d'un traitement antirétroviral (ART), d'un traitement préventif par cotrimoxazole (CPT), d'un dépistage de TB et d'un traitement préventif par isoniazide (IPT). L'analyse a mesuré les variations des caractéristiques des patients associées à l'offre de services.Résultats : Toutes les femmes ont bénéficié du HCT et 80% de celles ayant eu un test VIH positif ont eu un dépistage de TB. La majorité (85,9%) des femmes VIH-positives ont eu un comptage des CD4. Cependant, seulement 12,9% des femmes éligibles ont reçu une prophylaxie ART sur place ; seulement 35,7% ont été référées pour une mise en route de l'ART ; seulement 42.3% ont commencé l'IPT ; et aucune n'a reçu de CPT ni d'autres investigations relatives à la TB. Les femmes VIH négatives avaient 2,6 fois (IC95% 1,3­5,3) plus de chances de bénéficier d'un dépistage de TB que leurs homologues VIH positives.Conclusions: L'identification des femmes VIH positives et de celles suspectes de TB a été satisfaisante, mais la mise en œuvre des autres activités de collaboration TB-VIH a été insuffisante.

Marco de referencia: La prestación integrada de servicios de atención de la tuberculosis (TB) y la infección por el virus de la inmunodeficiencia humana (VIH) se recomienda ampliamente como parte de los servicios que se ofrecen a las madres durante la atención prenatal (ANC). En el presente estudio se evaluó la introducción de los servicios integrados de TB y VIH en una unidad hospitalaria de ANC.Método: Se examinaron las historias clínicas de una muestra aleatoria de 308 embarazadas que acudieron al servicio de ANC entre abril del 2011 y febrero del 2012. Se extrajeron datos de los registros y las historias clínicas de las pacientes. Los criterios de evaluación fueron la proporción de mujeres en quienes se practicó la orientación y las pruebas diagnósticas del VIH (HCT), el recuento de linfocitos CD4, el tratamiento antirretrovírico (ART), el tratamiento preventivo con cotrimoxazol (CPT), la detección sistemática de la TB y el tratamiento preventivo con isoniazida (IPT). En el análisis se midieron las variaciones en las características de las pacientes asociadas con la prestación de los servicios.Resultados: Todas las mujeres recibieron HCT y en 80% que obtuvieron un resultado positivo, se practicó la detección sistemática de la TB. En la mayoría de las pacientes positivas frente al VIH se practicó el recuento de linfocitos CD4 (85,9%). Sin embargo, solo 12,9% de las mujeres aptas recibieron la profilaxis ART en el lugar de la consulta; solo 35,7% se remitieron con el fin de comenzar el ART; apenas 42,3% de las pacientes comenzaron el IPT; y ninguna recibió CPT ni tuvo investigaciones complementarias por TB. Las mujeres con resultados negativos frente al VIH exhibieron un cociente de posibilidades 2,6 veces inferior de beneficiar de la detección sistemática de la TB, en comparación con las mujeres VIH positivas.Conclusión: Se constató una detección adecuada de las mujeres positivas frente al VIH y de casos con presunción clínica de TB, pero una prestación deficiente de las demás actividades de los servicios integrados del VIH y la TB.

Epidemiology of drug-resistant tuberculosis among children and adolescents in South Africa, 2005-2010.

OBJECTIVE: To describe the demographic and clinical characteristics of children and adolescents diagnosed with resistance to any anti-tuberculosis drug (drug-resistant tuberculosis; DR-TB) in South Africa. DESIGN: We retrospectively reviewed medical records of all children (<13 years) and adolescents (13 to <18 years) with DR-TB at specialty hospitals in four South African provinces from 2005 to 2010. RESULTS: During the review period, 774 children and adolescents (median age 11.3 years) were diagnosed with DR-TB at selected facilities. A high proportion of patients had a history of previous TB treatment (285/631; 45.2%), human immunodeficiency virus (HIV) infection (375/685; 54.7%), contact with a TB case (347/454; 76.4%), and smear-positive (443/729; 60.8%), cavitary (253/680, 38.7%) disease. Eighty-two per cent of patients with HIV infection received antiretroviral therapy. Of 626 patients diagnosed with multidrug-resistant TB (MDR-TB), 561 (89.6%) received a regimen consistent with national guidelines; the median length of treatment was 22 months (IQR 16-25). Among 400 patients with any DR-TB and a known outcome, 20.3% died during treatment. CONCLUSION: Pediatric DR-TB in these provinces is characterized by complex clinical features at diagnosis, with one in five children dying during treatment. History of previous treatment and contact with a TB patient indicate opportunities for earlier diagnosis and treatment to improve outcomes.

A partially protective clone from Cowdria ruminantium identified by using a Salmonella vaccine delivery system.

A strategy has been developed to screen the Cowdria genome, using a Salmonella vaccine delivery system, to identify genes that code for protection-stimulating proteins. We have cloned mini-libraries of Cowdria into this Salmonella system, used the recombinant bacteria to immunize outbred mice, and then challenged them after two weeks with a lethal dose of Cowdria. When one of these mini-libraries was tested in a group of 5 mice, one mouse lived much longer than the others. The experiment was repeated with each of the clones from the mini-library being tested individually in 10 mice, and one mouse survived the challenge. This clone has been tested repeatedly in larger groups of mice and is proven to protect 14% of outbred mice against a lethal Cowdria challenge.

Characterization of south african isolates of Salmonella enteritidis by phage typing, numerical analysis of RAPD-PCR banding patterns and plasmid profiles.

Eleven of the 33 strains of Salmonella enteritidis (S.E.) included in this study belonged to phage type 34. Six strains belonged to phage type 14, six strains to phage type 4 and four strains to phage type 7. The remaining six strains belonged to phage types 35, 1, 24var (a variation of phage type 24), 9a, 1b and an unknown phage type. The majority of S.E. phage type 34 strains (eight of the 11) grouped at R2 > or =0.45 into one RAPD-PCR cluster with two strains of phage types 4, a strain of phage type 24var and a strain of phage type 9a, indicating that they consist of a genetically heterogeneous collection of strains. Two of the remaining three phage type 34 strains grouped into two different clusters, well separated from the other phage type 34 strains. One strain of phage type 34 was genetically diverse and did not cluster with any of the strains included in this study. Three of the phage type 14 strains grouped into cluster 11 at R2 > or =0.72, suggesting that they are genetically closely related. However, the remaining three strains of phage type 14 grouped into two separate clusters. Strains of phage types 7, 35, and 1 grouped in one cluster at R2 > or = 0.55. Our results clearly indicated that S.E. strains of the same phage type are not always genetically related. On the other hand, strains of a high genetic relatedness classified as different phage types. No specific plasmid profile could be linked to any of the phage types. Based on results obtained by LD50 virulence tests, strains containing the 38 MDa plasmid are more virulent compared to strains which do not contain the plasmid.

Auxotrophic, plasmid-cured Salmonella enterica serovar typhimurium for use as a live vaccine in calves.

Calves were vaccinated orally, subcutaneously or intraperitoneally with a smooth, plasmid-cured strain of Salmonella enterica serovar typhimurium, strain 81. Oral vaccination was not effective, as only 1/5 calves survived challenge with virulent S. typhimurium. Strain 81 was attenuated for calves, as only a slight rise in rectal temperatures was detected after vaccination. The organism was excreted by some calves in the faeces, but no signs of diarrhoea were observed after vaccination. After parenteral vaccination, strain 81 was able to reach the intestines, gastric associated lymphoid tissues and other internal lymphoid tissues and remained viable for up to 14 days in the bovine host. After oral challenge with a virulent strain, 9/10 vaccinated calves survived challenge as opposed to 4/10 control calves (p<0.5). Diarrhoea was present in all calves of the control groups, but in only 4/10 of the vaccinated calves. The clinical reactions of the vaccinated calves were milder than in the control calves, as the rises in rectal temperatures were lower, diarrhoea was less severe, and the challenge strain was present in fewer organs from vaccinated calves than control calves. This study showed that parenterally administered Salmonella vaccines can induce both mucosal and systemic immunity, and it is postulated that this capability of strain 81 is related to its colonisation of lymphoid tissues and other systemic and intestinal tissues. This study confirmed that plasmid-cured strains were attenuated in the bovine host and conferred significant protection after parenteral vaccination, but not oral vaccination.

A comparative study of the growth of Campylobacter fetus strains in liquid media.

The growth of C. fetus fetus, C. fetus venerealis and C. fetus venerealis biotype intermedius was examined in 10 liquid media. From the data obtained, a 10% inoculum size and an oxygen level of 6% seemed imperative for consistent growth, especially for the C. fetus venerealis strain. A lowered redox potential obtained by the addition of 0, 1% cysteine-HC1 to the media was stimulatory. The medium which yielded the best growth was the one described by Dennis & Jones (1959). The fastidious C. fetus venerealis strain yielded maximum values of 0,5% packed cell volumes after 48 h cultivation in a microaerophilic atmosphere on this medium. The other strains yielded higher values.

The demonstration of the K-antigen of Campylobacter fetus venerealis using a microtitre agglutination test.

Using specific antisera prepared against heat-treated and untreated cells in a microtitre system, 7 strains of Campylobacter fetus venerealis were examined for the presence of K-antigen. The technique was effective for distinguishing between strains and it was found that 4 out of 7 isolates contained microcapsules, therefore being potentially suitable for vaccine production.

The production of an auxotrophic marked, plasmid-cured Salmonella ser. Typhimurium as a live attenuated vaccine.

A number of amino acid requiring auxotrophic strains of Salmonella Typhimurium were produced by chemical mutagenesis. One of them, strain 81, was cured of the virulence plasmid and attenuated for mice. This strain had an auxotrophic requirement for serine, which could be used as a marker for the differentiation of the vaccine strain from other isolates in the field. The strain still contained the smooth form of the O-antigen, was resistant to Complement-mediated killing of serum and produced type 1 fimbriae. Of the six auxotrophic mutants only this mutant differed in its outer membrane protein profile from that of the parent strain in that an outer membrane protein of about 30 kDa was absent. With the use of the polymerase chain reaction, using total DNA of the cell as template, and with primers targeted to the virulence plasmid, it was shown that the virulence plasmid of Salmonella Typhimurium was completely cured from this strain. This strain also had a LD50-value of 4 log units lower for mice than the parent strain. The plasmid-cured strain gave a very high degree of protection to mice after systemic immunization, but not after oral vaccination. Compared to the parent, strain 81 also had a lower multiplication rate in the liver and spleen after intraperitoneal inoculation, characteristics that could be attributed to plasmid-loss, and it could also not be recovered from the spleen and liver of orally inoculated mice.

The biochemical differentiation between Salmonella and Citrobacter.

A number of bacterial isolates which could not be identified as either Salmonella or Citrobacter by conventional biochemical tests and could not be typed as Salmonella with available antisera, were further examined biochemically and by lysis with phage Felix 0.1. Glycerol-positive salmonellae and lysine-positive citrobacters were encountered, which could be confused with the other genus, but when the reactions of such strains were examined in the other tests, accurate identifications could be done. Of the tests examined, glycerol fermentation, the beta-galactosidase test, lysine decarboxylation, sorbose fermentation, galacturonate fermentation and lysis by the phage could be used in the differentiation. These tests in combination, rather than 1 or 2 single tests gave reliable and conclusive differentiation.

Phage types of Salmonella enterica serovar Enteritidis isolated in South Africa from 1991-1995.

A total of 615 strains of Salmonella enterica serovar Enteritidis (SE), received from 1991-1995 at the Onderstepoort Veterinary Institute (OVI), were phage typed. Most SE isolates (54,7%) originated from poultry followed by humans (28,5 %) and poultry eggs (9,6 %). Phage type 34 was the most prevalent (40,5 %) of all isolates, followed by phage type 4 (33,8 %). Other phage types identified were 1, lb, 4a, 7, 7a, 9a, 14, 24, 24var and 35 (in total 2,4% of isolates). Most isolates of SE were received from the Western Cape Province (47,4%) and Gauteng (22,3%). In poultry phage type 4 was dominant, but in humans, eggs, goats, ducks, sheep, pigs and rabbits, phage type 34 was the dominant type. It appeared as if the poultry-associated epidemic of SE in South Africa that occurred from 1991-1995 originated in the Western Cape Province during 1991 amongst poultry and then spread from there to humans and eggs and then to the rest of the country, where it emerged during 1993. Results indicate that phage type 34 was the dominant phage type from 1991-1993, but during 1994-1995 its presence declined. During this latter period the presence of phage type 4 increased. This may suggest that two smaller epidemics consisting of the two different phage types might have been responsible for the epidemic that occurred from 1991-1995.

Salmonella isolated from crocodiles and other reptiles during the period 1985-1994 in South Africa.

Over a 10-year period, 173 isolates of Salmonella were obtained during routine isolation from reptiles. Of the 173 isolates, 92 different Salmonella serovars were identified. Of them, 61 (66%) belonged to subspecies I, nine to subspecies II and 21 to subspecies III (IIIa and IIIb), and one to subspecies IV. The majority of isolates were from farmed Nile crocodiles (145), three from wild-caught African dwarf crocodiles, 11 from captive snakes, 13 from lizards and one from a tortoise. The isolates from the tortoise and lizards were subspecies I isolates (Zaire and Tsevie, respectively). Of the snakes, nine isolates were S.III. The serovars isolated most often from the crocodiles were of subspecies I (32 serovars). Eight were from subspecies II, seven from subspecies III and one from subspecies IV. The most frequently identified serovars were Typhimurium (seven), Tsevie (six), Duval (six), Schwerin (six), Tinda (six), and Tallahassee (six). On two commercial crocodile breeding farms that had experienced ongoing problems for about two years, many isolates of Salmonella were made. Some of these serovars were isolated more than once, and also months apart. No single Salmonella serovar predominated, nor did a single pathological condition. These salmonellas were predominantly of subspecies I.

The isolation of Campylobacter hyointestinalis from a pig in South Africa.

The isolation of a strain of Campylobacter hyointestinalis from a piglet is described. The animal originated from a farm where another animal showed signs of intestinal adenomatosis. The animal from which the isolation was made had diarrhoea, and an enteropathogenic Escherichia coli was also isolated. No pathological changes indicative of intestinal adenomatosis were detected in this animal.

The first isolations of Campylobacter mucosalis from pigs in South Africa.

The first isolations of Campylobacter mucosalis in South Africa are described. Isolations were made from a 6-week-old weaner pig with necrotic enteritis and from 2 gingival swabs of suckling piglets from herds with histories of porcine intestinal adenomatosis. The isolates were serologically identified as being serotype A strains.