Browsing by Author "Moodley, Clinton"

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    A citywide, clonal outbreak of Pseudomonas aeruginosa during a drought
    (2021) Opperman, Christoffel Johannes; Centner, Chad M; Nicol , Mark P; Moodley, Clinton
    Background Outbreaks of community-acquired Pseudomonas aeruginosa are typically small and localized. We investigated an increase in P. aeruginosa clinical isolates in Cape Town, South Africa during a severe drought. Methods Cases were defined as P. aeruginosa isolated from any clinical sample, and “wild-type” as susceptibility to all antibiotics tested. Residential addresses of community-acquired wild-type cases were mapped. Whole genome sequencing and multi-locus sequence typing were used to determine clonality and identify virulence genes. A modified case-control study in a subset of patients with bloodstream infection compared demographic and clinical characteristics between sequence types. Results The outbreak lasted 10 months from December, 2016 to September, 2017 with 3,321 documented cases. At the peak, cases reached 2.3-fold baseline and the city's dams reached a nadir of 19% capacity. Cases were distributed widely across the city. Multi-locus sequence type (ST) 303 was found in 27 of 42 (64%) blood culture isolates of P. aeruginosa during the outbreak, one of 19 (5%) before, and none of 11 after. ST303 infection was independently associated with younger age, but not with co-morbidities nor increased mortality. Fifty-one virulence genes were differentially present in ST303 compared with other sequence types, including genes involved in biofilm formation, iron uptake, and gut penetration. Conclusion The investigation confirmed a citywide outbreak of P. aeruginosa coinciding with and potentially related to a severe drought. We identified a predominant outbreak-associated clone, ST303, which harboured genes that could contribute to virulence and survival in drought-related conditions. Enhanced surveillance for P. aeruginosa during periods of drought is recommended.
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    Diagnostic yield of tuberculosis investigations on bone marrow biopsy samples in HIV positive patients at Groote Schuur Hospital
    (2023) Baloyi, Xikombiso; Moodley, Clinton; Verburgh Estelle
    Background: Acid fast bacilli (AFB) staining on bone marrow samples has low sensitivity for diagnosing HIVassociated tuberculosis and Tuberculosis (TB) culture results may be delayed. The GeneXpert® MTB/RIF Ultra assay may provide a more sensitive diagnostic test on bone marrow biopsy samples. Methods: We conducted a two-stage study in a tertiary hospital in South Africa, initially assessing the retrospective yield of TB diagnoses on bone marrow biopsies in adult HIV-positive participants retrospectively from 01-01-2019 to 31-07-2020. Subsequently, determining the prospective yield and diagnostic performance of the GeneXpert® MTB/RIF Ultra assay on bone marrow aspirate and peripheral blood samples in adult HIV-positive participants undergoing bone marrow biopsy from 11- 08-2020 to 31-01-2021. Results: One hundred and twenty-two biopsies were analysed, of which 59/122 were performed for haematological malignancy staging. Granulomata with AFB were detected in six samples, and nine new lymphoma diagnoses were made. Bone marrow TB culture detected only one non-tuberculous mycobacterial infection. All 17 participants who had TB diagnosed from another clinical site were bone marrow TB culture negative. TB treatment was confirmed in 11/33 participants recruited prospectively. One trace positive GeneXpert® MTB/RIF Ultra result on peripheral blood was detected. All TB cultures on bone marrow aspirates and peripheral blood were negative. Conclusion: In a tertiary care hospital in South Africa, the utility of TB culture and GeneXpert® MTB/RIF Ultra on bone marrow aspirate specimens in HIV-positive participants was limited. We postulate that the initiation of empiric anti-tuberculosis treatment could have resulted in false negative results.
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    Molecular characterization of ABC-type multidrug efflux systems in Bifidobacterium longum subsp. longumT JCM 1217
    (2011) Moodley, Clinton; Reid, Sharon J; Abratt, Valerie Rose
    A healthy and stable gastrointestinal microbiota is a vital feature of the innate immune system. It affords the host numerous health benefits and acts as a barrier against opportunistic gut infections. Probiotic bacterial supplements are, therefore, widely used in industry to promote good health. There is, however, a need to understand not only the factors underlying the health promoting capabilities of these bacteria, but also the intrinsic antimicrobial resistance mechanisms which these bacteria are known to harbour. These antibiotic resistance traits confer a competitive advantage on these bacteria over other bacterial species where they reside in the gut. It also allows them to survive during antibiotic therapy and they are able to continue conferring health benefits on the host. To better understand the mechanisms these bacteria utilize in conferring antibiotic resistance, genes which confer multidrug resistance by the active hydrolysis of ATP were studied here. These genes belong to the ATP-binding cassette (ABC) family of efflux transporters.
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    Nasopharyngeal colonization dynamics with Streptococcus pneumoniae and associated antimicrobial resistance in a South African birth cohort
    (2019) Manenzhe, Rendani Innocent; Moodley, Clinton; Nicol, Mark; Dube, Felix
    Introduction: Nasopharyngeal (NP) colonization by Streptococcus pneumoniae (the pneumococcus) precedes the development of respiratory tract infection. Colonization by antimicrobial-resistant pneumococci, especially in infants, is a major public health concern as pneumococcus is a frequent cause of bacterial acute respiratory tract infections among children. This study longitudinally investigated antimicrobial resistance amongst pneumococci colonizing the nasopharynx of South African infants immunized with the 13- valent pneumococcal conjugate vaccine (PCV13). Furthermore, the study explored strainlevel pneumococcal colonization patterns and associated antimicrobial resistance determinants as well as the composition of the NP antibiotic resistome using shotgun metagenomic sequencing. Methods: NP swabs were collected every second week from birth through the first year of life from 137 infants who were immunized with 2+1 doses of PCV13. These were the first 137 infants enrolled in the cohort who had the most complete fortnightly NP sampling (defined as at least 23-26 fortnightly collected NP swabs). Pneumococci were identified and serotyped using conventional techniques, and their antibiotic susceptibility profiles determined by disc diffusion and E-test. A subset of 196 NP samples from 23 infants were selected based on changes in serotype or antimicrobial resistance. These were subjected to broth enrichment, total nucleic acid extraction and subsequent shotgun metagenomic sequencing. Sequence reads were assembled and aligned to reference pneumococcal genomes. In-silico pneumococcal capsular, multilocus sequence typing, and antimicrobial resistance determinants were described. Finally, antibiotic resistance genes were identified from all bacterial contigs, to determine the NP resistome. Results: 1520 pneumococcal (760 non-duplicate) isolates were recovered from 137 infants; including non-typeable (n = 99), PCV13 (n = 133), and non-PCV13 serotypes (n = 528). The prevalence of penicillin, erythromycin, and cotrimoxazole non-susceptibility was 19% (147/760; 95% CI 17-22%) (3% resistant), 18% (136/760; 95% CI 15-21%) (14% resistant) and 45% (344/760; 95% CI 42-49%) (36% resistant), respectively. The predominant penicillin-non-susceptible serotypes included 15B/15C (n = 20), 19A (n = 13), 15A (n = 10), 19F (n = 8), and 21 (n = 8). Multi-drug resistance (MDR) was observed in 9% (68/760; 95% CI 7-11%) of the isolates. PCV13 serotypes were more likely to be non-susceptible, compared to non-PCV13 serotypes, to penicillin (26% vs. 16%, p = 0.007), erythromycin (23% vs. 15%, p = 0.027) and cotrimoxazole (62% vs. 41%, p < 0.001). Non-susceptibility to penicillin, erythromycin, and cotrimoxazole remained relatively constant through the first year of life (X 2 test for trend: p = 0.184, range 0 – 25%; p = 0.171, range 0 – 27%; and p = 0.572, range 0 – 55%, respectively). Overall, penicillin or erythromycin-non-susceptible pneumococci were carried for a shorter duration than susceptible pneumococci (penicillin [mean days, 18 vs. 21, p = 0.013] and erythromycin [mean days, 18 vs. 21, p = 0.035]). Forty-five percentage (61/137) of infants carried the same serotype which acquired or lost resistance over time, and these changes were predominantly for penicillin (76%, 79/104). Of the 196 NP samples sequenced, 174 had corresponding positive cultures for pneumococci and, of these, 152 were assigned an in-silico serotype. Metagenomic sequencing detected a single pneumococcal serotype in 85% (129/152), and co-colonization in 15% (23/152) of NP samples, respectively. In total, 22 different pneumococcal serotypes were identified, with 15B/15C (n = 49) and 16F (n = 21) being the most common non-PCV13 serotypes, while 23F (n = 9) and 19A (n = 8) were the most common PCV13 serotypes. Twenty-six different sequence types (STs), including 4 novel STs were identified. Mutations in the folA and folP genes, associated with cotrimoxazole resistance, were detected in 89% (87/98) of cotrimoxazole-non-susceptible pneumococci and mutations in the pbp1a and pbp2x genes, known to confer beta-lactam resistance, were identified in penicillin nonsusceptible ST705215B/15C isolates. A total of 329 antimicrobial resistance (AMR) genes were detected in 64% (125/196) of the sequenced samples, including 36 non-redundant genes ranging from 1 to 14 genes per sample. The predominant AMR genes detected were those conferring resistance to beta-lactams (52%, 172/329), macrolide-lincosamide-streptogramin (17%, 56/329), and tetracycline (12%, 38/329). The msrD, ermB, and mefA genes were only detected from pneumococcal reads. The predominant resistance genes detected from nonpneumococcal reads included blaOXA-60, blaOXA-22, and blaBRO-1. Conclusion: NP carriage of antibiotic-non-susceptible pneumococci was relatively constant throughout the first year of life. Despite high vaccine coverage levels, PCV13 serotypes were identified and were more commonly non-susceptible to penicillin, erythromycin, and cotrimoxazole. Overall, penicillin or erythromycin-non-susceptible pneumococci were carried for a shorter duration than susceptible pneumococci, however, non-susceptible PCV13 serotypes were carried for a longer duration than non-susceptible non-PCV13 serotypes. Direct shotgun sequencing from enriched NP samples was shown to be a powerful technique for a detailed description of the pneumococcal component of the NP microbiome and resistome, and its use should be explored similarly for other bacteria in this niche.
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    Passive surveillance of STI pathogens in Cape Town, South Africa: A six-month molecular epidemiology study
    (2023) Moodley, Clinton; Engel, Mark
    Background: Sexually transmitted infections are among the most commonly occurring globally, with countries in sub-Saharan Africa exhibiting disproportionately higher prevalence rates. Reports indicate the need for accurate detection, epidemiological characterisation, and appropriate management of these infections. This prospective passive surveillance study sought to document local STI prevalence and to evaluate the potential of a molecular assay as a surveillance tool in our setting. Methods: Urogenital swabs, submitted to Groote Schuur Hospital over a period of 6 months, for routine microbiological investigations, were subjected to a commercial multiplex PCR assay to determine the distribution of STI pathogens. Correlations between detected organisms and clinical and demographic information were determined using Stata® software. Results: A total of 148 urogenital swabs were collected and tested, with the majority from women. Up to 83.79% of the samples tested positive for one or more pathogen, with all seven assayed pathogens detected in one or more sample. Ureaplasma parvum was the most prevalent pathogen detected overall, with a 6-month period prevalence of 42.57%, followed by N. gonorrhoeae (37.84%), M. hominis (34.46%), U. urealyticum (23.65%), T. vaginalis (11.49%), C. trachomatis (10.14%), with M. genitalium (1.35%) the least prevalent. There were several different combinations of co-infections with multiple pathogens, with one sample testing positive for five organisms. M. hominis and T. vaginalis were only detected in co-infection with other pathogens. Persons aged 17-30- and 31-40- years old were 51-times and 16-times, respectively, more likely to test PCR-positive for one or more STI pathogen. Samples submitted with non-urogenital specific indications were 11.82 times more likely to test positive for C. trachomatis. There was an association between samples submitted for GBS screening and PCR-positivity for any of the pathogens tested, which were 3.03 times more likely to test positive for U. parvum. Routine microbiological investigations only detected three infections. Conclusions: There is a significantly higher than expected rate and difference in organism distribution of STI prevalence in Cape Town, South Africa as compared with global and regional estimates. The use of molecular testing methods may improve detection, providing rapid results, which may allow for tailored guidelines and interventions to limit spread and resistance.
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    Technical evaluation of a Real-time polymerase chain reaction (PCR) assay for the detection of Bartonella spp for diagnostic purposes
    (2022) Booley, Ghowa; Paul, Lynthia; Moodley, Clinton; Naicker, Preneshni
    Infective endocarditis (IE) is a rare disease affecting heart tissues. The laboratory diagnosis of culture-negative endocarditis is complicated, and largely based on the combination of nucleic acid detection methods and serological investigation. There is a paucity of published data on microbes causing culture-negative endocarditis, but a recent report indicated that the bacterium Bartonella was the commonest cause of culture-negative endocarditis at a tertiary care facility in Cape Town, South Africa. This laboratory-based, non-clinical pilot study, evaluated the utility of a previously published real-time PCR assay for detecting Bartonella spp. on cats. This will be the first time this target will be evaluated in a real-time PCR assay to detect Bartonella spp. in human samples. For this study, we constructed a plasmid vector containing an insert of 83bp, derived from the Bartonella nuoG gene. In this non-clinical, laboratory evaluation, we used one laboratory sample to amplify the nuoG bacterial DNA fragment and cloned it into a plasmid vector. Using this plasmid in a technical validation, we demonstrated that the previously described assay could detect nuoG when using the LightCycler 480 Probes Master Mix. The results indicated that the assay reliably detected as little as 1000 copies of the target DNA, and infrequently also detected 10-100 copies of the target. The study showed no amplification using some commonly encountered organisms found in our clinical setting, thus indicating 100% specificity for Bartonella. We demonstrated that a plasmid construct containing an internal fragment from the nuoG gene successfully detected the target using a real-time PCR assay. Future testing should include further optimisation to improve reaction efficiency of the assay with spiked diagnostic samples, including peripheral blood, and DNA extracted from heart valve samples. The utility of the RT-PCR for diagnostic purposes should be evaluated by comparing assay turnaround time, sensitivity, and specificity of this assay versus the conventional PCR and Sanger sequencing currently in use to detect Bartonella spp. in heart valves. We concluded that the assay exhibited strong potential for use as a diagnostic PCR using the constructed plasmid, but that further optimization to improve PCR efficiency, and work to determine the clinical sensitivity and specificity are needed before the assay can be applied to blood samples.
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    The genomic characterization of carbapenemase-producing Serratia marcescens at a tertiary hospital in South Africa
    (2023) Overmeyer, Amanda; Moodley, Clinton
    Background Serratia marcescens is an opportunistic nosocomial pathogen, and recent reports have highlighted the rapid increase in multidrug resistance in this organism. There is a paucity in genomic data for carbapenem resistant S. marcescens (CRSM). Methods A retrospective cohort study describing laboratory confirmed CRSM from a tertiary academic hospital in Cape Town, South Africa, for the period 2015-2020, was performed. Stored CRSM and control isolates were submitted for whole-genome sequencing using Illumina MiSeq, with the Nextera DNA Flex Library Preparation Kit. Sequence data was analysed in-house using srst2 and Tychus, and CRSM and control isolates were compared. Results Twenty-one CRSM and four control isolates were sequenced and analysed. Twenty-four different resistance genes were identified, with all isolates having at least two resistance genes, and seventeen isolates harbouring three or more genes. This correlated well with phenotypic results. The blaOXA-48-like carbapenemase was the most common carbapenemase identified in 86% (18/21) of CRSM. A core SNP difference tree indicated that the CRSM could be grouped into three clusters. A minority of isolates had shared plasmids. Several genes and single nucleotide polymorphisms (SNPs) were identified in the CRSM which may putatively augment virulence, but this requires further functional characterisation. Conclusion A diverse resistome was observed in CRSM, which was also reflected phenotypically, with blaOXA-48-like the most common carbapenemase. Though distinct clusters were observed, no clonality was noted, and a limited number of isolates shared plasmids. This study provides genomic data for emerging CRSM and highlights the importance of ongoing genomic surveillance to inform infection prevention control and antimicrobial stewardship initiatives.
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    The Pneumococcus Urinary Antigen Test Kit: Use in the laboratory for the presumptive diagnosis of pneumococcal bacteraemia
    (2020) Tootla, Hafsah Deepa; Bamford, Colleen; Moodley, Clinton
    Introduction: Culture remains the ‘gold standard' for diagnosis of Streptococcus pneumoniae bacteraemia. Time to definitive identification using culture is 24–48 hours, and prior antibiotic therapy, the ability of S. pneumoniae to self-autolyse and its fastidious nature can yield no growth on culture. Novel detection methods for invasive pneumococcal disease include PCR and antigen tests. We evaluated using a urine antigen test directly on selected blood cultures with appropriate Gram stain results, immediately after signalling positive for the rapid identification of S. pneumoniae bacteraemia. Method: We collected 212 blood cultures that had signalled positive with an automated blood culture system, and then yielded gram-positive cocci in pairs/chains or cocci with uncertain morphological arrangement. The BinaxNOW Streptococcus pneumoniae urinary antigen test, routine culture with optochin and real time lytA PCR was performed on all samples. Diagnostic accuracy analysis (sensitivity and specificity) of the antigen test and Gram stain with gram-positive cocci in pairs was each compared to culture positivity for S. pneumoniae, PCR positivity and the composite of culture or PCR positivity for S. pneumoniae as the reference standards. Results: S. pneumoniae (Spn) was cultured in 55 samples, gram-positive organisms other than S. pneumoniae (NSpn) in 140 samples and 17 samples had no growth (NG). Grampositive cocci in pairs was predominant on Gram stain in the Spn/NG groups whilst the minority in the NSpn group. In the Spn group, all except 1 sample which was antigen positive but PCR negative, were antigen and PCR positive. In the NSpn group, antigen and PCR was negative in 123 samples, antigen and PCR positive in 1 sample and antigen positive but PCR negative in the remaining 16 samples. In the NG group, antigen and PCR were positive in 16 samples and antigen positive but PCR negative in 1 sample. Sensitivity of the antigen test compared to culture, PCR or the composite of culture or PCR was 100%. Specificity was 87-88% but increased to 93-96% when used in subsets with gram-positive cocci in pairs or clinical history compatible with respiratory illness or meningitis. Sensitivity and specificity of the antigen test when compared to Gram stain using gram-positive cocci in pairs (69%-75% and 81% respectively) were both higher. Discussion and Conclusion: Accurate and rapid diagnosis of S. pneumoniae bacteraemia is challenging with current diagnostic tools. Specificity of the antigen test is mostly limited by crossreactivity with viridans streptococci, coagulase negative staphylococci and enterococcus species, but this can be overcome if Gram stain morphology and clinical history is available. Sensitivity and specificity of Gram stain alone in predicting S. pneumoniae bacteraemia is poor and is increased with use of the antigen test. The antigen test is a useful adjunctive tool improving diagnosis and turnaround time of S. pneumoniae bacteraemia. In settings like ours, where high-level resistance, defined as minimum inhibitory concentration ≥2μg/mL to penicillin is still relatively low (~7%), rapid de-escalation to penicillin in the appropriate clinical setting would be possible with the introduction of such test and could also potentially be a suitable alternative to molecular testing for S. pneumoniae identification in samples with no growth on culture.
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    The utility of a real-time PCR to detect Leptospira in a routine diagnostic setting
    (2022) Chanda, Raphael; Paul, Lynthia; Moodley, Clinton; Naicker, Preneshni
    Leptospirosis is a neglected zoonotic infection with world-wide distribution. A paucity of leptospirosis data from the African continent exists, mainly due to limited access to diagnostics. The clinical presentation ranges from mild to severe disease with multi organ involvement, while the mild form mimics another common tropical disease i.e., malaria. The gold standard for diagnostic detection currently is an immunological test discerning the presence of specific antibodies present in the immune phase of the disease. The serological methods are hindered by the inability to distinguish past from current infection and utility limited to only the immune phase of the disease. Due to lack of sensitivity and specificity in serological methods, improved diagnostic methods are needed to aid early identification in the acute phase. Methods should also distinguish saprophyte and pathogenic species. To address this gap, we developed an inhouse PCR assay targeting the microbe's rrs and lipL32 genes, using primer sets previously reported in literature to be both sensitive and specific for pathogenic Leptospira spp. Using inhouse constructed plasmids, we did a non-clinical, technical validation employing probe-based, real time polymerase chain reaction assays and a locally available commercial kit. Although our assay needs further optimization, we demonstrated that the PCR reliably detected 100 copies and 1000 copies of Leptospira rrs and lipL32 targets respectively. To test specificity, we did real-time PCR with pure DNA from a selected set of pathogens known to be prevalent in bacteremia's in local settings and observed that the rrs target was amplified with Group B streptococci as template but no other tested pathogens, while no non-specific amplification for lipL32 was observed. The non-specific amplification had been reported previously in the literature, suggesting the rrs gene is not a good target to use, even when primers are specifically designed to only detect Leptospira rrs. Future work using the assay should include optimizing assay performance using DNA extracted from the ideal clinical samples to detect Leptospira, namely urine and blood of patients clinically suspected to have leptospirosis. However, the assay demonstrated potential for use as a diagnostic PCR using the constructed plasmid, but further optimization to improve PCR efficiency and assessing its performance in clinical setting is required
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    Utility of the biofire filmarray pneumonia panel plus assay for syndromic testing of lower-respiratory tract infections in a low-middle-income setting
    (2025) Van Der Westhuyzen, Mene; Moodley, Clinton
    Background: Determining lower respiratory tract infection (LRTI) aetiology is complex. Culture-based methods are laborious with poor sensitivity. Molecular assays improve detection of potential pathogens, but incorrect interpretation of results may lead to inappropriate antimicrobial therapy. Methods: The utility of the BioFire® FilmArray® Pneumonia Panel plus (FA-PP) to detect LRTI pathogens, and the potential antimicrobial stewardship impacts in a low resource setting, was assessed. Routine LRT samples were included from adult patients with clinically suspected LRTI or with a concomitant blood culture at Groote Schuur Hospital and referring facilities. Culture and FA-PP results were compared, and pharmacy data analysed to determine appropriateness of antibiotic therapy. Results: There was an 80% correlation between cultured LRTI pathogens and the FA-PP bin ≥107 results. Compared to culture the FA-PP detected substantially more pathogens (86,6% versus 17,9%) and produced a combined 100% positive percent agreement, and 88% negative percent agreement. The FA-PP detected bacterial-viral coinfections in 27% of samples. Correlation of FA-PP results with pharmacy data (n=69) indicated a potential antibiotic change in 75% of cases, but this is difficult to accurately characterise without a ‘gold-standard' for treatment or complete clinical data. Conclusion: The FA-PP increased the number of positive samples with typical bacteria, but the semi-quantitative reporting algorithm does not describe the correlation between the different bin values and colonization versus infection. This complicates result interpretation and may lead to inappropriate antimicrobial treatment. This study highlights the potential positive impact of rapid molecular assays for routine care in lower income settings, but also underscores the interpretive challenges associated with these tests.
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    Validation of a Pan-fungal polymerase chain reaction (PCR) assay for the detection and identification of medically important fungi in a diagnostic laboratory
    (2018) Ntuli, Sindile Venessa; Moodley, Clinton; Bamford, Colleen
    The laboratory diagnosis of fungal infection is complicated, based on the microscopic detection, culture isolation, and detection of serological response. In recent years, there has been an increase in the utilization of molecular diagnostic techniques for the detection and accurate identification of fungal pathogens. This was a laboratory accuracy study evaluating the performance of a selected pan-fungal PCR using 70 previously identified reference fungal isolates. The DNA yield and purity of three different DNA extraction methods was assessed, using 6 representative fungal isolates. The ZR Fungal/Bacterial DNA MicroPrep™ produced a median concentration of 17.28 ng/μl,which was significantly higher (p value = 0.0079) than the MagNA pure LC DNA Isolation Kit III and QIAamp DNA Mini Kit, which produced median yields of 11.08ng/μl and 3.54 ng/μl, respectively. The selected pan-fungal PCR was optimized PCR and successfully performed on 62 of the 70 reference isolates. A selection of 56 amplicons were submitted for DNA sequence determination. Of all the sequences queried on the NCBI and Ribosomal Development Project (RDP) databases, 95/111(86%) were concordant with the results obtained from the reference laboratory. Study findings have shown that the selected pan-fungal PCR, coupled with DNA sequence analysis is an excellent diagnostic tool for the identification of medically relevant fungi. This assay, in combination with conventional culture, is useful for the rapid and accurate identification of fungal isolates. Future work will involve evaluating the utility of this assay for the detection and identification of medically relevant fungi in deep tissue biopsies.