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In 2020, the Namibian Ministry of Health and Social Services (MoHSS) and the Robert Koch Institute (RKI) started a twinning project with the long-term goal of establishing a Namibia Institute of Public Health (NIPH). A fundamental pillar of an NIPH is a fully operational Public Health laboratory system. Due to the COVID-19 pandemic, the need for strengthening the existing Namibian Laboratory system became eminent. Following the Intra-Action Review (IAR) of the COVID-19 response in Namibia in 2020, three regional diagnostic laboratories, at points of entry, were assessed. The major issues identified were long delays between sampling of both suspected cases and COVID-19 patients and receiving test results due to extended sample transport times to the central laboratory in Windhoek and the overload of the central capacities due to overwhelming numbers of samples during peak times. This led to the establishment of three SARS-CoV-2 PCR diagnostic laboratories through procurement and installation of equipment, provision of consumables/reagents, and on-site training of laboratory technicians with continued virtual technical support. Consequently, an important reduction of the diagnosis turnaround time was achieved. Of great value was the technical support given by the staff at the central laboratory during the trainings allowing for immediate validation of the newly established laboratories and to strengthen the communication between regional laboratories and the central laboratory. The Namibian molecular diagnostic capacities have increased in important regions in Namibia and will provide data to support the health policies of the future NIPH. New diagnostic protocols will be developed to foster the sustainability of the established laboratories and could support the implementation of genomic surveillance capacities. Finally, stronger relationships were built through these joint activities, which will support and the next steps of the establishment of the NIPH. Key messages • Supporting and Strengthening the Namibian Public Health Laboratory system. • Long-term goal of establishing a Namibia Institute of Public Health (NIPH).

Bacillus anthracis spores have been shown to be an efficient biological weapon and their recent use in bioterrorist attacks has demonstrated the need for rapid and specific diagnostics. A TaqMan real-time PCR for identification of B. anthracis was developed, based on the two plasmids, pX01 and pX02, both of which are necessary for pathogenicity, as well as on the chromosomally encoded rpoB gene. Bacteria picked from colonies or pelleted from liquid cultures were directly inoculated into the PCR mix, thus avoiding time-consuming DNA preparation and minimizing handling risks. B. anthracis spores were cultivated for a few hours in enrichment broth before PCR analysis, or used directly for real-time PCR, thus allowing to confirm or exclude potential attacks approximately 2-3 h after the material has arrived in the laboratory.

A real-time quantitative polymerase chain reaction assay was devised to determine the load of human herpesvirus (HHV)-6A and -6B DNA in paired samples of plasma and peripheral blood leukocytes (PBL) of 25 bone marrow transplant patients. The assay detects HHV-6 DNA variants A and B in a linear range of 10(7)-10(1) genome equivalents per assay. Viral DNA was measured in 336 paired DNA PBL samples and in corresponding plasma samples. HHV-6A and/or -6B DNA was detected in PBL of 23 of 25 patients and in plasma of 24 of 25 patients. HHV-6B was the predominant variant found in PBL and also was detected in the corresponding plasma. Surprisingly, only 1 of 25 patients had detectable HHV-6A DNA in PBL, although 23 of 25 patients were positive for HHV-6A DNA in plasma. HHV-6 DNA load in plasma was significantly higher for HHV-6A than for HHV-6B (P=.0066).

The use of common reverse transcription (RT)-PCR reference target sequences can produce false-positive results by amplification of either contaminating DNA or processed pseudogenes. Furthermore, qualitative RT-PCR alone cannot distinguish between high- and poor-quality cDNA preparations, which again may be crucial for the interpretation of low-abundance transcripts. We have developed a highly sensitive quantitative RT-PCR for β-actin, using the TaqManTM chemistry. Through this technique, we were able to quantitatively detect 10 β-actin molecules per 100 ng of cDNA without coamplification of pseudogenes or genomic DNA. Thus, the presented method may be advantageous for the interpretation of quantitative RT-PCR results. PCR analysis may be difficult to interpret unless a reference target sequence is amplified in parallel. This control reaction is necessary to evaluate whether a sufficient amount of amplifiable material is present in the sample investigated. Thus, negative PCR can be defined as such only when amplification of a reference gene reveals a positive result. Amplifying a reference is especially important in RT-PCR because RNA can be degraded rapidly before or during cDNA synthesis. As reference sequences for RT-PCR, so called “housekeeping” genes are preferred because they are constitutionally expressed by all cell types. β-Actin is an attractive candidate for reference coamplification because it exhibits only minor intraindividual kinetic changes and is not primarily affected by any human disease (1). However, a major concern with β-actin and other commonly used references such as glyceraldehyde-3-phosphate dehydrogenase is that processed pseudogenes can be coamplified by primers that are originally restricted to cDNA. This problem is difficult to circumvent because sequence homologies of >90% exist between mRNA and the respective pseudogenes (2); thus, the pseudogenes usually cannot be distinguished in gel electrophoresis. Furthermore, as long as genomic DNA contamination cannot safely be excluded, DNA may be coamplified together with cDNA. Thus, positive analysis of …

Background: Ebola virus (EBOV) RNA persistence in semen, reported sexual transmission, and sporadic clusters at the end of the 2013-2016 epidemic have prompted recommendations that male survivors refrain from unprotected sex unless their semen is confirmed to be EBOV free. However, there is no fully validated assay for EBOV detection in fluids other than blood. Methods: The Cepheid Xpert Ebola assay for EBOV RNA detection was validated for whole semen and blood using samples obtained from uninfected donors and spiked with inactivated EBOV. The validation procedure incorporated standards from Clinical and Laboratory Standards Institute and Good Clinical Laboratory Practices guidelines for evaluating molecular devices for use in infectious disease testing. Results: The assay produced limits of detection of 1000 copies/mL in semen and 275 copies/mL in blood. Limits of detection for both semen and blood increased with longer intervals between collection and testing, with acceptable results obtained up to 72 hours after specimen collection. Conclusions: The Cepheid Xpert Ebola assay is accurate and precise for detecting EBOV in whole semen. A validated assay for EBOV RNA detection in semen informs the care of male survivors of Ebola, as well as recommendations for public health.

Penicillin-binding proteins (PBPs) from penicillin-susceptible strains of Streptococcus pneumoniae are believed to be fairly similar in contrast to PBPs occurring in resistant isolates. The antigenic variation of PBPs 1a and 2b in 65 penicillin-susceptible strains from different geographic areas and a wide variety of isolation sites was analyzed using a set of specific antisera and monoclonal antibodies. Three strains contained different antigenic variants of PBP 1a, and 50 strains contained one of three antigenic variants found in PBP 2b. Seven patterns of antibody reactivity could be defined; all but one were distinct from those found recently in resistant strains. In addition, electrophoretic mobilities of all six PBPs, compared after conventional SDS-PAGE and fluorography, revealed an unexpected variation of PBP-profiles even for strains of one sero-group. Few strains appeared identical to each other or to the laboratory reference strain R6.

A 17-year-old girl developed a cutaneous macular lesion in the area of the right sternoclavicular joint. In the 3-week period after initial symptoms were noted, the lesion passed through papular, vesicular, and pustular stages before forming a hard black eschar (diameter, 2 cm) with erythema and edema, as well as regional lymphangitis (figure 1). In the early stage of the disease, the patient experienced malaise and had subfebrile temperatures. After treatment with several different antibiotic regimens (clindamycin-clarithromycin, then fucidine-imipeFigure 2. Hard black eschar with erythema and edema in the area of the sternoclavicular joint.

Antisera raised against penicillin-binding protein (PBP) 1a of Streptococcus pneumoniae reacted with PBP 2 in certain strains of Streptococcus pyogenes. Cross-reactivity could be demonstrated on immunoblots as well as by immunoprecipitation of native solubilised proteins, indicating a similar structural arrangement also in the native form of the two PBPs.