Preparing healthcare facilities in sub-Saharan Africa for future outbreaks: insights from a multi-country digital self-assessment of COVID-19 preparedness.

BACKGROUND: Despite previous experience with epidemics, African healthcare systems were inadequately prepared and substantially impacted by the coronavirus disease 2019 (COVID-19) pandemic. Limited information about the level of COVID-19 preparedness of healthcare facilities in Africa hampers policy decision-making to fight future outbreaks in the region, while maintaining essential healthcare services running. METHODS: Between May-November 2020, we performed a survey study with SafeCare4Covid - a free digital self-assessment application - to evaluate the COVID-19 preparedness of healthcare facilities in Africa following World Health Organization guidelines. The tool assessed (i) COVID-19-related capabilities with 31 questions; and (ii) availability of essential medical supplies with a 23-supplies checklist. Tailored quality improvement plans were provided after assessments. Information about facilities' location, type, and ownership was also collected. RESULTS: Four hundred seventy-one facilities in 11 African countries completed the capability assessment; 412 also completed the supplies checklist. The average capability score on a scale of 0-100 (n=471) was 58.0 (interquartile range 40.0-76.0), and the average supplies score (n=412) was 61.6 (39.0-83.0). Both scores were significantly lower in rural (capability score, mean 53.6 [95%CI:50.3-57.0]/supplies score, 59.1 [55.5-62.8]) versus urban facilities (capability score, 65.2 [61.7-68.7]/supplies score, 70.7 [67.2-74.1]) (P<0.0001 for both comparisons). Likewise, lower scores were found for public versus private clinics, and for primary healthcare centres versus hospitals. Guidelines for triage and isolation, clinical management of COVID-19, staff mental support, and contact tracing forms were largely missing. Handwashing stations were partially equipped in 33% of facilities. The most missing medical supply was COVID-19 specimen collection material (71%), while 43% of facilities did not have N95/FFP2 respirators and 19% lacked medical masks. CONCLUSIONS: A large proportion of public and private African facilities providing basic healthcare in rural areas, lacked fundamental COVID-19-related capabilities and life-saving personal protective equipment. Decentralization of epidemic preparedness efforts in these settings is warranted to protect healthcare workers and patients alike in future epidemics. Digital tools are of great value to timely measure and improve epidemic preparedness of healthcare facilities, inform decision-making, create a more stakeholder-broad approach and increase health-system resilience for future disease outbreaks.

Short communication: high rates of thymidine analogue mutations and dual-class resistance among HIV-infected Ugandan children failing first-line antiretroviral therapy.

HIV-infected children are at high risk of acquiring drug-resistant viruses, which is of particular concern in settings where antiretroviral drug options are limited. We aimed to assess resistance patterns and predict viral drug susceptibility among children with first-line antiretroviral therapy (ART) failure in Uganda. A cross-sectional analysis of children switching ART regimens due to first-line failure was performed at three clinical sites in Uganda. HIV-RNA determination and genotypic resistance testing on all specimens with HIV-RNA >1,000 copies/ml were performed. Major drug resistance mutations were scored using the 2011 International Antiviral Society-USA list. The Stanford algorithm was used to predict drug susceptibility. At the time of switch, 44 genotypic resistance tests were available for 50 children. All children harbored virus with nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance [95% confidence interval (CI) 92-100%] and NRTI resistance was present in 98% (95% CI 88-100%). Forty-six percent (95% CI 30-61%) of children harbored ≥2 thymidine analog mutations. M184V was identified as the only NRTI mutation in 27% (95% CI 15-43%). HIV susceptibility to NRTIs, with the exception of tenofovir, was reduced in ≥60% of children. Ugandan children experiencing first-line ART failure in our study harbored high rates of dual-class and accumulated HIV drug resistance. Methods to prevent treatment failure, including adequate pediatric formulations and alternative second-line treatment options, are urgently needed.

Identification of sequential viral escape mutants associated with altered T-cell responses in a human immunodeficiency virus type 1-infected individual.

Control of viremia in natural human immunodeficiency virus type 1 (HIV-1) infection in humans is associated with a virus-specific T-cell response. However, still much is unknown with regard to the extent of CD8(+) cytotoxic T-lymphocyte (CTL) responses required to successfully control HIV-1 infection and to what extent CTL epitope escape can account for rises in viral load and ultimate progression to disease. In this study, we chose to monitor through full-length genome sequence of replication-competent biological clones the modifications that occurred within predicted CTL epitopes and to identify whether the alterations resulted in epitope escape from CTL recognition. From an extensive analysis of 59 biological HIV-1 clones generated over a period of 4 years from a single individual in whom the viral load was observed to rise, we identified the locations in the genome of five CD8(+) CTL epitopes. Fixed mutations were identified within the p17, gp120, gp41, Nef, and reverse transcriptase genes. Using a gamma interferon ELIspot assay, we identified for four of the five epitopes with fixed mutations a complete loss of T-cell reactivity against the wild-type epitope and a partial loss of reactivity against the mutant epitope. These results demonstrate the sequential accumulation of CTL escape in a patient during disease progression, indicating that multiple combinations of T-cell epitopes are required to control viremia.

Primary effect of chemotherapy on the transcription profile of AIDS-related Kaposi's sarcoma.

BACKGROUND: Drugs & used in anticancer chemotherapy have severe effects upon the cellular transcription and replication machinery. From in vitro studies it has become clear that these drugs can affect specific genes, as well as have an effect upon the total transcriptome. METHODS: Total mRNA from two skin lesions from a single AIDS-KS patient was analyzed with the SAGE (Serial Analysis of Gene Expression) technique to assess changes in the transcriptome induced by chemotherapy. SAGE libraries were constructed from material obtained 24 (KS-24) and 48 (KS-48) hrs after combination therapy with bleomycin, doxorubicin and vincristine. KS-24 and KS-48 were compared to SAGE libraries of untreated AIDS-KS, and to libraries generated from normal skin and from isolated CD4+ T-cells, using the programs USAGE and HTM. SAGE libraries were also compared with the SAGEmap database. RESULTS: In order to assess the primary response of AIDS-related Kaposi's sarcoma (AIDS-KS) to chemotherapy in vivo, we analyzed the transcriptome of AIDS-KS skin lesions from a HIV-1 seropositive patient at two time points after therapy. The mRNA profile was found to have changed dramatically within 24 hours after drug treatment. There was an almost complete absence of transcripts highly expressed in AIDS-KS, probably due to a transcription block. Analysis of KS-24 suggested that mRNA pool used in its construction originated from poly(A) binding protein (PABP) mRNP complexes, which are probably located in nuclear structures known as interchromatin granule clusters (IGCs). IGCs are known to fuse after transcription inhibition, probably affecting poly(A)+RNA distribution.Forty-eight hours after chemotherapy, mRNA isolated from the lesion was largely derived from infiltrating lymphocytes, confirming the transcriptional block in the AIDS-KS tissue. CONCLUSIONS: These in vivo findings indicate that the effect of anti-cancer drugs is likely to be more global than up- or downregulation of specific genes, at least in this single patient with AIDS-KS. The SAGE results obtained 24 hrs after chemotherapy can be most plausibly explained by the isolation of a fraction of more stable poly(A)+RNA.

Phylogenetic relationships among the species of the genus Testudo (Testudines: Testudinidae) inferred from mitochondrial 12S rRNA gene sequences.

To test phylogenetic relationships within the genus Testudo (Testudines: Testudinidae), we have sequenced a fragment of the mitochondrial (mt) 12S rRNA gene of 98 tortoise specimens belonging to the genera Testudo, Indotestudo, and Geochelone. Maximum likelihood and neighbor-joining methods identify two main clades of Mediterranean tortoises, one composed of the species Testudo graeca, Testudo marginata, and Testudo kleinmanni and a second of Testudo hermanni, Testudo horsfieldii, and Indotestudo elongata. The first clade, but not the second, was also supported by maximum parsimony analysis. Together with the genus Geochelone, a star-like radiation of these clades was suggested, as a sister-group relationship between the two Testudo clades could not be confirmed. The intraspecies genetic variation was examined by sequencing the mt 12S rRNA fragment from 28 specimens of T. graeca and 49 specimens of T. hermanni from various geographic locations. Haplotype diversity was found to be significantly larger in T. graeca compared with T. hermanni, suggestive of reduced genetic diversity in the latter species, perhaps due to Pleistocene glaciations affecting northern and middle Europe or other sources of lineage reduction. No ancient mt 12S rRNA gene haplotypes were identified in T. graeca and/or T. hermanni originating from islands in the Mediterranean Sea, suggesting that these islands harbor tortoise populations introduced from the European and African mainland.

Routine DNA analysis based on 12S rRNA gene sequencing as a tool in the management of captive primates.

Automated DNA sequencing of a fragment of the relatively slowly evolving mitochondrial 12S rRNA gene was used to distinguish primate species, and the method was compared with species determination based upon classical taxonomy. DNA from blood from 53 monkeys housed at the Stichting AAP Shelter for Exotic Animals, all Old World monkeys, was amplified by polymerase chain reaction (PCR) with a primer set spanning approximately 390 nucleotides of the mitochondrial 12S rRNA gene. The products were directly sequenced and compared with our database of primate 12S sequences. Many individuals were found to harbor a 12S sequence identical to one of the reference sequences. For others, phylogenetic methods were used for species estimation, which was especially informative in Cercopithecus species.

St. Kitts green monkeys originate from West Africa: Genetic evidence from feces.

Sequencing of a fragment of mitochondrial DNA extracted from droppings of a green monkey inhabiting the Caribbean island of St. Kitts, and comparing the obtained sequence with sequences determined earlier for the four recognized subspecies of African green monkeys, showed that this monkey can be classified as Cercopithecus aethiops sabaeus, and thus originates from West Africa. As the ancestors of the monkeys reached the island by ships involved in the slave trade in the 17th to 18th centuries, determination of the monkey subspecies suggests that the animals were originally acquired nearby the West African ports from which the ships sailed, and were not brought from the central parts of Africa together with the slaves. © 1996 Wiley-Liss, Inc.