Prospective Cohort Study of Treatment Outcomes of Vaginal Discharge Syndrome in Women in Windhoek, Namibia.

BACKGROUND: Syndromic treatment is the standard of care for vaginal discharge syndrome (VDS) in resource-constrained settings. However, the outcomes of VDS treatment have not been well documented. This study aimed to determine the incidence, risk factors, and microbial etiology of treatment failure in women with VDS. METHODS: This prospective cohort study of women with VDS was conducted between September 2021 and March 2022 at Katutura Intermediate Hospital in Windhoek, Namibia. Microbiological analyses of sexually transmitted infections (STIs; Chlamydia trachomatis , Neisseria gonorrhoeae , Trichomonas vaginalis , Mycoplasma genitalium ), bacterial vaginosis, and vulvovaginal candidiasis (VVC) were performed. Treatment outcomes were assessed at 7 and 30 days after treatment, followed by microbial investigation in case of treatment failure. RESULTS: One hundred nine women were enrolled, and 94 (86%) completed the follow-up. At baseline, 58 of 109 women (53%) were diagnosed with STI, 47 of 109 (43%) with bacterial vaginosis, and 45 of 109 (41%) with VVC. Candida albicans (33 of 45; 73%) was the main pathogen in VVC, with fluconazole resistance detected in 8 of 33 isolates (24%); 10 of 12 (80%) of non- albicans Candida species showed resistance. The incidence of treatment failure was 3.6 per 100 person-years at 7 days and 1.0 per 100 person-years at 30 days of follow-up; 17 of 94 women (18%) had recurrent VDS, and 12 of 94 women (13%) had persistent VDS. Vulvovaginal candidiasis (odds ratio, 4.3; 95% confidence interval, 1.7-11; P = 0.002) at baseline was associated with treatment failure. CONCLUSIONS: Treatment failure after syndromic management of VDS is common in resource-constrained settings. Access to diagnostic testing, including fungal culture and susceptibility testing, is recommended to improve outcomes.

Genomic epidemiology and molecular characteristics of blaNDM-1-positive carbapenem-resistant Pseudomonas aeruginosa belonging to international high-risk clone ST773 in the Gauteng region, South Africa.

PURPOSE: The emergence of carbapenem-resistant P. aeruginosa (CRPA) harbouring acquired carbapenemase genes (blaVIM, blaIMP and blaNDM) has become a global public health threat. Three CRPA isolates included in the study had an extensively drug-resistant phenotype with susceptibility to colistin only and were positive for the blaNDM-1 gene. The current study aimed to investigate the genomic epidemiology and molecular characteristics of the blaNDM-1-positive CRPA isolates collected from the Gauteng region, South Africa. METHODS: Short read whole genome sequencing (WGS) was performed to determine sequence types (STs), genetic relatedness, resistome, virulome and the genetic environment of the blaNDM-1 gene. RESULTS: The WGS and phylogenetic analyses revealed that the study isolates belonged to an international high-risk clone ST773 and belonged to the same clade with eight blaNDM-1-positive ST773 isolates from Hungary, India, Nigeria, South Korea and USA. The study isolates harboured a wide repertoire of intrinsic and acquired antibiotic resistance genes (ARGs) related with mobile genetic elements, porins and efflux pumps, as well as virulence factor genes. The clade-specific ARGs (blaNDM-1, floR2/cmlA9, rmtB4, tetG) were found in a putative integrative and conjugative element (ICE) region similar to ICE6660-like. CONCLUSION: As ICE carrying the blaNDM-1 gene can easily spread to other P. aeruginosa isolates and other Gram-negative bacteria, the findings in this study highlight the need for appropriate management strategies and active surveillance of CRPA isolates in the Gauteng region, South Africa.

Molecular epidemiology and antimicrobial resistance of vaginal Candida glabrata isolates in Namibia.

Candida glabrata is the most common non-albicans Candida species that causes vulvovaginal candidiasis (VVC). Given the intrinsically low susceptibility of C. glabrata to azole drugs, investigations into C. glabrata prevalence, fungal susceptibility profile, and molecular epidemiology are necessary to optimise the treatment of VVC. This molecular epidemiological study was conducted to determine antifungal drug profile, single nucleotide polymorphisms (SNPs) associated with phenotypic antifungal resistance and epidemic diversity of C. glabrata isolates from women with VVC in Namibia. Candida glabrata isolates were identified using phenotypic and molecular methods. Antifungal susceptibility of strains was determined for fluconazole, itraconazole, amphotericin B, and anidulafungin. Whole genome sequencing was used to determine SNPs in antifungal resistance genes and sequence type (ST) allocation. Among C. glabrata isolates, all (20/20; 100%) exhibited phenotypic resistance to the azole class antifungal drug, (fluconazole), and phenotypic susceptibility to the polyene class (amphotericin B), and the echinocandins (anidulafungin). Non-synonymous SNPs were identified in antifungal resistance genes of all fluconazole-resistant C. glabrata isolates including ERG6 (15%), ERG7 (15%), CgCDR1 (25%), CgPDR1 (60%), SNQ2 (10%), FKS1 (5.0%), FKS2 (5.0%), CgFPS1 (5.0%), and MSH2 (15%). ST15 (n = 8/20, 40%) was predominant. This study provides important insight into phenotypic and genotypic antifungal resistance across C. glabrata isolates from women with VVC in Namibia. In this study, azole resistance is determined by an extensive range of SNPs, while the observed polyene and echinocandin resistance-associated SNPs despite phenotypic susceptibility require further investigation.

Candida glabrata is inherently resistant to azole drugs. In this study, we identified a clone that was predominant in women with vulvovaginal candidiasis in Namibia, and that harboured various mutations in resistance-associated genes. This study provides important insight into antifungal resistance across C. glabrata isolates in a sub-Sahara African setting.

Salmonella enterica serovar Typhi H58 clone has been endemic in Zimbabwe from 2012 to 2019.

BACKGROUND: Typhoid fever, caused by S. enterica ser. Typhi, continues to be a substantial health burden in developing countries. Little is known of the genotypic diversity of S. enterica ser. Typhi in Zimbabwe, but this is key for understanding the emergence and spread of this pathogen and devising interventions for its control. OBJECTIVES: To report the molecular epidemiology of S. enterica ser. Typhi outbreak strains circulating from 2012 to 2019 in Zimbabwe, using comparative genomics. METHODS: A review of typhoid cases records from 2012 to 2019 in Zimbabwe was performed. The phylogenetic relationship of outbreak isolates from 2012 to 2019 and emergence of antibiotic resistance was investigated by whole-genome sequence analysis. RESULTS: A total 22 479 suspected typhoid cases, 760 confirmed cases were reported from 2012 to 2019 and 29 isolates were sequenced. The majority of the sequenced isolates were predicted to confer resistance to aminoglycosides, ß-lactams, phenicols, sulphonamides, tetracycline and fluoroquinolones (including qnrS detection). The qnrS1 gene was associated with an IncN (subtype PST3) plasmid in 79% of the isolates. Whole-genome SNP analysis, SNP-based haplotyping and resistance determinant analysis showed that 93% of the isolates belonged to a single clade represented by multidrug-resistant H58 lineage I (4.3.1.1), with a maximum pair-wise distance of 22 SNPs. CONCLUSIONS: This study has provided detailed genotypic characterization of the outbreak strain, identified as S. Typhi 4.3.1.1 (H58). The strain has reduced susceptibility to ciprofloxacin due to qnrS carried by an IncN (subtype PST3) plasmid resulting from ongoing evolution to full resistance.

Molecular epidemiological analysis of Mycoplasma genitalium shows low prevalence of azithromycin resistance and a well-established epidemic in South Africa.

OBJECTIVES: Macrolide resistance in Mycoplasma genitalium is emerging globally. There is paucity of data from sub-Saharan Africa where syndromic management is used to treat sexually transmitted infections (STIs). We conducted a molecular epidemiological study to determine the prevalence of azithromycin resistance and epidemic diversity of M. genitalium infections in South Africa. METHODS: We analysed 90 M. genitalium-positive specimens that had been collected consecutively from men and women (50% symptomatic) from geographically diverse communities across the northern part of South Africa between 2015 and 2019. Melting curve analysis followed by targeted sequencing of the 23S rRNA gene was performed to detect azithromycin resistance. Molecular typing was done through single nucleotide polymorphism (SNP) analysis of the MG191 gene and short tandem repeats (STR) assessment of the MG309 gene. An overview of all published M. genitalium sequence types was generated and novel sequence types identified in this study were allocated numbers accordingly. RESULTS: Azithromycin resistance was detected in 1/90 M. genitalium-positive specimens (1.1%; 95% CI 0% to 3.3%) as conferred by A2071G mutation; this strain also harboured a C234T mutation in the parC gene with wild type gyrA gene. SNP typing and STR assessment was successful in 38/90 specimens (42%) and showed a genetically diverse epidemic, without geographic clustering, with eight novel sequence types identified. CONCLUSION: This is the first study that determines resistance in M. genitalium infection since introduction of azithromycin in the syndromic management regimen for STIs in South Africa in 2015. Despite a well-established epidemic, azithromycin-resistant M. genitalium infection is still uncommon in the public healthcare sector. However, it has the potential to undermine the effectiveness of syndromic management. Introduction of molecular diagnostics and continuous surveillance are warranted for early detection emergence of resistance.

Single-Dose Azithromycin for Genital Lymphogranuloma Venereum Biovar Chlamydia trachomatis Infection in HIV-Infected Women in South Africa: An Observational Study.

ABSTRACT: We conducted an observational study of lymphogranuloma venereum (LGV) biovar Chlamydia trachomatis infection in HIV-infected women in South Africa. The LGV biovar was detected in vaginal specimens of 17 (20%) of 85 women with C. trachomatis infection; 29% were symptomatic. All cases were negative for the LGV biovar after single-dose azithromycin.

Molecular epidemiology of extended-spectrum beta-lactamase-producing extra-intestinal pathogenic Escherichia coli strains over a 2-year period (2017-2019) from Zimbabwe.

This study was designed to characterize extended-spectrum beta-lactamase (ESBL)-producing extra-intestinal pathogenic Escherichia coli (E.coli) (ExPEC) associated with urinary tract infections in nine different geographic regions of Zimbabwe over a 2-year period (2017-2019). A total of 48 ESBL-positive isolates from urine specimen were selected for whole-genome sequencing from 1246 Escherichia coli isolates biobanked at the National Microbiology Reference laboratory using phenotypic susceptibility testing results from the National Escherichia coli Surveillance Programme to provide representation of different geographical regions and year of isolation. The majority of ESBL E. coli isolates produced cefotaximase-Munich (CTX-M)-15, CTX-M-27, and CTX-M-14. In this study, sequence types (ST) 131 and ST410 were the most predominant antimicrobial-resistant clones and responsible for the increase in ESBL-producing E. coli strains since 2017. Novel ST131 complex strains were recorded during the period 2017 to 2018, thus showing the establishment and evolution of this antimicrobial-resistant ESBL clone in Zimbabwe posing an important public health threat. Incompatibility group F plasmids were predominant among ST131 and ST410 isolates with the following replicons recorded most frequently: F1:A2:B20 (9/19, 47%), F2:A1: B (5/19, 26%), and F1:A1:B49 (8/13, 62%). The results indicate the need for continuous tracking of different ESBL ExPEC clones on a global scale, while targeting specific STs (e.g. ST131 and ST410) through control programs will substantially decrease the spread of ESBLs among ExPEC.

The Global Ascendency of OXA-48-Type Carbapenemases.

Surveillance studies have shown that OXA-48-like carbapenemases are the most common carbapenemases in Enterobacterales in certain regions of the world and are being introduced on a regular basis into regions of nonendemicity, where they are responsible for nosocomial outbreaks. OXA-48, OXA-181, OXA-232, OXA-204, OXA-162, and OXA-244, in that order, are the most common enzymes identified among the OXA-48-like carbapenemase group. OXA-48 is associated with different Tn1999 variants on IncL plasmids and is endemic in North Africa and the Middle East. OXA-162 and OXA-244 are derivatives of OXA-48 and are present in Europe. OXA-181 and OXA-232 are associated with ISEcp1, Tn2013 on ColE2, and IncX3 types of plasmids and are endemic in the Indian subcontinent (e.g., India, Bangladesh, Pakistan, and Sri Lanka) and certain sub-Saharan African countries. Overall, clonal dissemination plays a minor role in the spread of OXA-48-like carbapenemases, but certain high-risk clones (e.g., Klebsiella pneumoniae sequence type 147 [ST147], ST307, ST15, and ST14 and Escherichia coli ST38 and ST410) have been associated with the global dispersion of OXA-48, OXA-181, OXA-232, and OXA-204. Chromosomal integration of blaOXA-48 within Tn6237 occurred among E. coli ST38 isolates, especially in the United Kingdom. The detection of Enterobacterales with OXA-48-like enzymes using phenotypic methods has improved recently but remains challenging for clinical laboratories in regions of nonendemicity. Identification of the specific type of OXA-48-like enzyme requires sequencing of the corresponding genes. Bacteria (especially K. pneumoniae and E. coli) with blaOXA-48, blaOXA-181, and blaOXA-232 are emerging in different parts of the world and are most likely underreported due to problems with the laboratory detection of these enzymes. The medical community should be aware of the looming threat that is posed by bacteria with OXA-48-like carbapenemases.

Antimicrobial Resistance of Neisseria gonorrhoeae Isolates from High-Risk Men in Johannesburg, South Africa.

Neisseria gonorrhoeae antimicrobial drug resistance has emerged worldwide; however, the situation in sub-Saharan Africa is not well documented. We investigated the molecular epidemiology and occurrence of antimicrobial resistance in Neisseria gonorrhoeae infections in two core transmission groups of men in Johannesburg, South Africa. We recruited men who have sex with men (MSM) presenting with urethral discharge and men with recurrent episodes of urethral discharge. Molecular testing and culture for N. gonorrhoeae were performed, followed by antimicrobial susceptibility testing. Whole-genome sequencing (WGS) was used to identify resistance-conferring mutations and to determine the genetic relatedness of the isolates. In all, 51 men were recruited; 42 (82%) had N. gonorrhoeae infections. Most gonococcal isolates were resistant to ciprofloxacin (78%) and tetracycline (74%); 33% were penicillin resistant. All gonococcal isolates were susceptible to cephalosporins and spectinomycin. Azithromycin resistance was observed in 4 (15%) isolates (epidemiological cutoff), all with mutations in the mtrR promoter region. Most of the isolates (19/27) harbored the gonococcal genetic island, which is associated with antimicrobial resistance. WGS revealed a diverse epidemic with mostly novel NG-STAR (70%) and NG-MAST (70%) sequence types. Thus, we demonstrate a high prevalence of antimicrobial resistance in Neisseria gonorrhoeae strains obtained from high-risk men in South Africa. The introduction of diagnostics and scale-up of surveillance are warranted to prevent the emergence of multidrug-resistant infections.

Klebsiella pneumoniae ST307 with blaOXA-181, South Africa, 2014-2016.

Klebsiella pneumoniae sequence type (ST) 307 is an emerging global antimicrobial drug-resistant clone. We used whole-genome sequencing and PCR to characterize K. pneumoniae ST307 with oxacillinase (OXA) 181 carbapenemase across several private hospitals in South Africa during 2014-2016. The South Africa ST307 belonged to a different clade (clade VI) with unique genomic characteristics when compared with global ST307 (clades I-V). Bayesian evolution analysis showed that clade VI emerged around March 2013 in Gauteng Province, South Africa, and then evolved during 2014 into 2 distinct lineages. K. pneumoniae ST307 clade VI with OXA-181 disseminated over a 15-month period within 42 hospitals in 23 cities across 6 northeastern provinces, affecting 350 patients. The rapid expansion of ST307 was most likely due to intrahospital, interhospital, intercity, and interprovince movements of patients. This study highlights the importance of molecular surveillance for tracking emerging antimicrobial clones.

Provision of Sexually Transmitted Infection Services in a Mobile Clinic Reveals High Unmet Need in Remote Areas of South Africa: A Cross-sectional Study.

BACKGROUND: The burden of sexually transmitted infections (STIs) in areas of sub-Saharan Africa with poor access to health care services is not well documented. In remote areas of South Africa, we investigated the prevalence of STIs and approaches to providing STI services through a mobile clinic. METHODS: We recruited 251 adult women visiting a mobile clinic that normally provides general health education and screening services, but not STI care. Clinical and sexual history was obtained and vaginal specimens were tested for Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, and Mycoplasma genitalium infection and for Candida albicans and bacterial vaginosis. RESULTS: Laboratory test was positive for 133 (53%) of 251 women for at least 1 STI: C. trachomatis was observed in 52 (21%) women, N. gonorrhoeae in 39 (16%) women, T. vaginalis in 81 (32%) women and M. genitalium in 21 (8%) women. Eighty-one (32%) women met the criteria for vaginal discharge syndrome, of which 58% (47/81) would have been treated accurately. Among asymptomatic women 84 (49%) of 170 were diagnosed with an STI but untreated under the syndromic approach. We could not identify factors associated with asymptomatic STI infection. CONCLUSIONS: There is a high unmet need for STI care in rural South African settings with poor access to health care services. Provision of STI services in a mobile clinic using the syndromic management approach provides a useful approach, but would have to be enhanced by targeted diagnostics to successfully address the burden of infection.

High Rate of Repeat Sexually Transmitted Diseases Among Men Who Have Sex With Men in South Africa: A Prospective Cohort Study.

We observed a high rate of incident sexually transmitted diseases within 55 days (median) of follow-up (78% retention) among symptomatic men who have sex with men (n = 78) in South Africa (188 per 100 person-years; 95% confidence interval, 1.2-2.7); 16 newly acquired and 10 with persistent positivity. This highlights the need to strengthen prevention efforts, whereas introduction of diagnostics is urgently warranted.

A rare case of Colistin-resistant Salmonella Enteritidis meningitis in an HIV-seropositive patient.

BACKGROUND: Non-typhoidal salmonellae (NTS) have been associated with invasive disease, notably meningitis, in immunocompromised individuals. Infections of this nature carry high rates of morbidity and mortality. Colistin resistance in salmonellae is a rare finding, more so in an invasive isolate such as cerebrospinal fluid (CSF). Colistin resistance has important infection control implications and failure to manage this phenomenon may lead to the loss of our last line of defence against multi-drug resistant Gram-negative organisms. To our knowledge, this is the first reported clinical case of colistin-resistant Salmonella Enteritidis meningitis in South Africa. CASE PRESENTATION: We report a case of a young male patient with advanced human immunodeficiency virus (HIV) infection who presented to hospital with symptoms of meningitis. Cerebrospinal fluid (CSF) cultured a Salmonella Enteritidis strain. Antimicrobial susceptibility testing (AST) of the isolate, revealed the strain to be colistin resistant. Despite early and aggressive antimicrobial therapy, the patient succumbed to the illness after a short stay in hospital. Subsequent genomic analysis of the isolate showed no presence of the mcr genes or resistance-conferring mutations in phoPQ, pmrAB, pmrHFIJKLME/arnBCADTEF, mgrB, and acrAB genes, suggesting the presence of a novel colistin resistance mechanism. CONCLUSION: Invasive non-typhoidal salmonellae infection should be suspected in patients with advanced immunosuppression who present with clinical features of meningitis. Despite early and appropriate empiric therapy, these infections are commonly associated with adverse outcomes to the patient. Combination therapy with two active anti-Salmonella agents may be a consideration in the future to overcome the high mortality associated with NTS meningitis. Colistin resistance in clinical Salmonella isolates, although a rare finding at present, has significant public health and infection control implications. The causative mechanism of resistance should be sought in all cases.

Prevalence and Detection of Trichomonas vaginalis in HIV-Infected Pregnant Women.

BACKGROUND: Trichomonas vaginalis is a sexually transmitted infection associated with increased transmission of HIV and significant adverse birth outcomes; culture and polymerase chain reaction (PCR) are commonly used in diagnosis. METHODS: Consenting HIV-infected pregnant women were recruited from clinics in South Africa and screened for T. vaginalis using PCR. Polymerase chain reaction-positive women provided an additional sample for culture. We compared T. vaginalis detection between PCR and culture, and investigated how PCR cycle threshold (Ct) values differ among culture results. RESULTS: A total of 359 women were enrolled and 76 (20%) tested T. vaginalis PCR positive. Cultures were obtained from 61 of the PCR-positive women, and 38 (62%) were culture positive. The median baseline Ct of the PCR-positive/culture-positive group was 22.6 versus 38.0 among those who were PCR positive/culture negative (P < 0.001). Culture-positive cases had lower Ct values (higher DNA load); a Ct value less than 30 predicted positivity with a sensitivity of 97% and a specificity of 96%. CONCLUSIONS: Culture was positive in roughly half of PCR-positive cases. The culture-negative cases had significantly higher Ct values, indicating a lower concentration of T. vaginalis DNA. A Ct value of 30 provides a reliable threshold for predicting culture positivity. The clinical significance of culture-negative infections detected by PCR is still unclear.

Identification and characterization of Staphylococcus devriesei isolates from bovine intramammary infections in KwaZulu-Natal, South Africa.

BACKGROUND: Coagulase-negative staphylococci (CoNS) are among the leading bacterial causes of bovine mastitis in many dairy-producing countries. Among the challenges associated with the specific diagnosis of CoNS infections is the biochemical heterogeneity of the species in the genus and the unavailability of accurate, cost-effective and up-to-date diagnostic tests. A previous study investigating the diversity of CoNS associated with cases of bovine mastitis in South Africa, resulted in six CoNS isolates which could not be identified despite the use of a combination of different molecular assays. The identification and characterisation of the isolates was pursued further in this study. RESULTS: The six CoNS isolates in question were identified by sequencing multiple housekeeping genes (dnaJ, hsp60, rpoB, 16S rRNA) and characterized through the use of matrix-assisted laser/desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and the Biolog GEN III Microplate™ bacterial identification system. Sequencing of housekeeping genes identified the isolates as S. devriesei. This Staphylococcus species was only described in 2010 and this is the first report documenting the isolation of S. devriesei from cases of bovine IMIs in South Africa. Analysis of mass spectra generated by the six isolates showed intra-species variation which was also observed when evaluating the metabolic profiles of the isolates using the Biolog GEN III system. Neither the MALDI-TOF MS nor the Biolog database are currently populated with data relating to S. devriesei, resulting in the isolates not being identified, in the case of MALDI-TOF MS analysis, or mis-identified as was observed with the Biolog GEN III system. CONCLUSIONS: The phenotyping data collected during this investigation provides useful information concerning Staphylococcus devriesei which could be used to populate user system databases thereby ensuring the accurate identification of isolates in future. The availability of improved diagnostics will in turn facilitate studies to elucidate the epidemiology, pathogenicity and true prevalence of this species in dairy herds.

Chlamydia trachomatis Biovar L2 Infection in Women in South Africa.

We detected Chlamydia trachomatis biovar L2 in vaginal swab specimens of 7 women with vaginal discharge in South Africa. Whole-genome sequencing directly from clinical specimens identified a closely related cluster of strains. The clinical role of this infection in the context of syndromic management should be clarified.

Etiology of bacterial vaginosis and polymicrobial biofilm formation.

Microorganisms in nature rarely exist in a planktonic form, but in the form of biofilms. Biofilms have been identified as the cause of many chronic and persistent infections and have been implicated in the etiology of bacterial vaginosis (BV). Bacterial vaginosis is the most common form of vaginal infection in women of reproductive age. Similar to other biofilm infections, BV biofilms protect the BV-related bacteria against antibiotics and cause recurrent BV. In this review, an overview of BV-related bacteria, conceptual models and the stages involved in the polymicrobial BV biofilm formation will be discussed.

Normal flora and bacterial vaginosis in pregnancy: an overview.

The female genital tract is an intricate, yet balanced ecosystem that hosts a variety of different residential microflora. The physiological changes that occur during pregnancy may disrupt this balanced ecosystem and predispose women to a potentially pathogenic microbiota. Bacteria that are associated with bacterial vaginosis (BV) are opportunistic pathogens that frequently form part of this microbiota. The overgrowth of and infections with these bacteria are linked to poor obstetric outcomes and increased transmission of other reproductive tract infections (RTIs). These infections increase women's susceptibility of acquiring HIV, the rates of HIV shedding and the development of acquired immune deficiency syndrome (AIDS) in HIV-infected patients. It is unknown how the plethora of bacterial species associated with BV contributes to the dynamics of this condition. The use of high-throughput methods have led to the in-depth investigation of different BV-related bacterial species and the functional capabilities of these species. However, the pathogenesis of BV is still poorly defined and the role of individual BV-related bacterial species in specific pregnancy complications is unclear and controversial. The majority of BV infections are asymptomatic and successful diagnosis is complicated by the lack of reliable and standardized diagnostic tests.