School of Pathology (Journal Articles)

Permanent URI for this collectionhttps://hdl.handle.net/10539/37877

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Start date: 2014End date: 2019

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    Performance of the Biomark HD real-time qPCR System (Fluidigm) for the detection of nasopharyngeal bacterial pathogens and Streptococcus pneumoniae typing
    (Nature Research, 2019-04) Olwagen, Courtney P.; Adrian, Peter V.; Madhi, Shabir A.
    Traditional qPCR assays for pneumococcal detection and serotype characterization require large sample volume, is expensive and labor intensive. We aimed to develop a quantitative nanofuidic Fluidigm assay to overcome some of these shortcomings. A quantitative Fluidigm assay was established to detect 11 bacterial pathogens, 55 pneumococcal serotypes and 6 serotypes of H. infuenzae. The Fluidigm assay results were compared to conventional qPCR and culture. All reactions in the Fluidigm assay efectively amplifed their respective targets with high sensitivity and specifcity compared to qPCR. There was excellent concordance between qPCR and Fluidigm for detection of carriage prevalence (kappa>0.75) and density (Rho>0.95). Fluidigm identifed an additional 7 (4.2%) serotypes over those detected by qPCR. There was a modest concordance between culture and Fluidigm for the majority of reactions detecting S. pneumoniae serotypes/serogroups (kappa>0.6), with Fluidigm identifying an additional 113 (39.1%) serotypes. Discordant results between the three methods were associated with a low carriage density. The Fluidigm assay was able to detect common pneumococcal serotypes, H. infuenzae serotypes, and other common nasopharyngeal bacterial organisms simultaneously. Deployment of this assay in epidemiological studies could provide better insight into the efect of PCV immunization on the nasopharyngeal microbiota in the community
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    Characterization of putative DD-carboxypeptidase-encoding genes in Mycobacterium smegmatis
    (Nature Research, 2019-03) Ealand, Christopher S.; Asmal, Rukaya; Mashigo, Lethabo; Campbell, Lisa; Kana, Bavesh D.
    Penicillin binding proteins (PBPs) are the target of numerous antimicrobial agents that disrupt bacterial cell wall synthesis. In mycobacteria, cell elongation occurs through insertion of nascent cell wall material in the sub-polar region, a process largely driven by High Molecular Weight PBPs. In contrast, the function of DD-carboxypeptidases (DD-CPases), which are Low Molecular Weight Class 1C PBPs, in mycobacteria remains poorly understood. Mycobacterium smegmatis encodes four putative DD-CPase homologues, which display homology to counterparts in Escherichia coli. Herein, we demonstrate that these are expressed in varying abundance during growth. Deletion of MSMEG_1661, MSMEG_2433 or MSMEG_2432, individually resulted in no defects in growth, cell morphology, drug susceptibility or spatial incorporation of new peptidoglycan. In contrast, deletion of MSMEG_6113 (dacB) was only possible in a merodiploid strain expressing the homologous M. tuberculosis operon encoding Rv3627c (dacB), Rv3626c, Rv3625c (mesJ) and Rv3624c (hpt), suggestive of essentiality. To investigate the role of this operon in mycobacterial growth, we depleted gene expression using anhydrotetracyclineresponsive repressors and noted reduced bipolar peptidoglycan synthesis. These data point to a possible role for this four gene operon, which is highly conserved across all mycobacterial species, in regulating spatial localization of peptidoglycan synthesis.
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    Evaluation of automated malaria diagnosis using the Sysmex XN-30 analyser in a clinical setting
    (BioMed Central, 2019-01) Pillay, Evashin; Bezuidenhout, Belinda C.; Coetzer, Thérèsa L.; Khodaiji, Shanaz; Litshie, Monwabisi
    Background: Early and accurate diagnosis of malaria is a critical aspect of eforts to control the disease, and several diagnostic tools are available. Microscopic assessment of a peripheral blood smear enables direct visualization of parasites in infected red blood cells and is the clinical diagnostic gold standard. However, it is subjective and requires a high level of skill. Numerous indirect detection methods are in use, but are not ideal since surrogate markers of infection are measured. This study describes the frst clinical performance evaluation of the automated Sysmex XN-30 analyser, which utilizes fuorescence fow cytometry to directly detect and quantitate parasite-infected red blood cells. Results: Residual EDTA blood samples from suspected malaria cases referred for routine diagnosis were analysed on the XN-30. Parasitaemia was reported as a percentage, as well as absolute numbers of infected red blood cells, and scattergrams provided a visual image of the parasitized red blood cell clusters. The results reported by the XN-30 correlated with microscopy and the analyser demonstrated 100% sensitivity and specifcity. Measurements were reproducible and storage of samples at room temperature did not afect the parameters. Several Plasmodium species were detected, including Plasmodium falciparum, Plasmodium vivax and Plasmodium ovale. The XN-30 also identifed the transmissible gametocytes as a separate cluster on the scattergrams. Abnormal red blood cell indices (low haemoglobin and raised reticulocyte counts), haemoglobinopathies and thrombocytopenia did not interfere with the detection of parasites. The XN-30 also generated a concurrent full blood count for each sample. Conclusions: The novel technology of the Sysmex XN-30 provides a robust, rapid, automated and accurate platform for diagnosing malaria in a clinical setting. The objective enumeration of red blood cells infected with Plasmodium species makes it suitable for global use and allows monitoring of the parasite load once therapy has been initiated, thereby providing an early marker of drug resistance. The automated generation of a full blood count for each sample provides an opportunity for detecting unsuspected cases. Asymptomatic carriers can also be identifed, which will be useful in blood transfusion centres, and will enable treatment of these individuals to prevent the spread of the disease.
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    The importance of morphological identification of African anopheline mosquitoes (Diptera: Culicidae) for malaria control programmes
    (BMC, 2018-01) Erlank, Erica; Koekemoer, Lizette L.; Coetzee, Maureen
    Background: The correct identification of disease vectors is the first step towards implementing an effective control programme. Traditionally, for malaria control, this was based on the morphological differences observed in the adults and larvae between different mosquito species. However, the discovery of species complexes meant that genetic tools were needed to separate the sibling species and today there are standard molecular techniques that are used to identify the two major malaria vector groups of mosquitoes. On the assumption that species-diagnostic DNA polymerase chain reaction (PCR) assays are highly species-specific, experiments were conducted to investigate what would happen if non-vector species were randomly included in the molecular assays. Methods: Morphological keys for the Afrotropical Anophelinae were used to provide the a priori identifications. All mosquito specimens were then subjected to the standard PCR assays for members of the Anopheles gambiae complex and Anopheles funestus group. Results: One hundred and fifty mosquitoes belonging to 11 morphological species were processed. Three species (Anopheles pretoriensis, Anopheles rufipes and Anopheles rhodesiensis) amplified members of the An. funestus group and four species (An. pretoriensis, An. rufipes, Anopheles listeri and Anopheles squamosus) amplified members of the An. gambiae complex. Conclusions: Morphological identification of mosquitoes prior to PCR assays not only saves time and money in the laboratory, but also ensures that data received by malaria vector control programmes are useful for targeting the major vectors.
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    Molecular and physiological analysis of Anopheles funestus swarms in Nchelenge, Zambia
    (BMC, 2018-01) Zawada, Jacek W.; Dahan‑Moss, Yael L.; Muleba, Mbanga; Dabire, Roch K.; Maïga, Hamid; Venter, Nelius; Davies, Craig; Hunt, Richard H.; Coetzee, Maureen; Koekemoer, Lizette L.
    Background: Anopheles funestus has been recognized as a major malaria vector in Africa for over 100 years, but knowledge on many aspects of the biology of this species is still lacking. Anopheles funestus, as with most other anophelines, mate through swarming. A key event that is crucial for the An. funestus male to mate is genitalia rotation. This involves the 135° to 180° rotation of claspers, which are tipped with claws. This physical change then enables the male to grasp the female during copulation. The aim of this investigation was to molecularly characterize wild An. funestus swarms from Zambia and examine the degree of genitalia rotation within the swarm. Methods: Anopheles funestus swarms were collected from Nchelenge, northern Zambia, during dusk periods in May 2016. All the adults from the swarm were analysed morphologically and identified to species level using a multiplex PCR assay. Anopheles funestus s.s. specimens were molecularly characterized by restriction fragment length polymorphism type and Clade type assays. The different stages of genitalia rotation were examined in the adult males. Results: A total of six swarms were observed during the study period and between 6 and 26 mosquitoes were caught from each swarm. Species analysis revealed that 90% of the males from the swarms were An. funestus s.s. MWtype, with 84% belonging to clade I compared to 14% clade II and 2% failed to amplify. Very few specimens (3.4%) were identified as Anopheles gambiae s.s. Eighty percent of the males from the swarm had complete genitalia rotation. Conclusions: This is the first time that An. funestus swarms have been molecularly identified to species level. Anopheles funestus swarms appear to be species-specific with no evidence of clade-type differentiation within these swarms. The An. funestus swarms consist mainly of males with fully rotated genitalia, which strongly suggests that swarming behaviour is triggered primarily when males have matured.
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    The importance of morphological identification of African anopheline mosquitoes (Diptera: Culicidae) for malaria control programmes
    (BioMed Central (BMC), 2018-01) Erlank, Erica; Koekemoer, Lizette L.; Coetzee, Maureen
    Background: The correct identification of disease vectors is the first step towards implementing an effective control programme. Traditionally, for malaria control, this was based on the morphological differences observed in the adults and larvae between different mosquito species. However, the discovery of species complexes meant that genetic tools were needed to separate the sibling species and today there are standard molecular techniques that are used to identify the two major malaria vector groups of mosquitoes. On the assumption that species-diagnostic DNA polymerase chain reaction (PCR) assays are highly species-specific, experiments were conducted to investigate what would happen if non-vector species were randomly included in the molecular assays. Methods: Morphological keys for the Afrotropical Anophelinae were used to provide the a priori identifications. All mosquito specimens were then subjected to the standard PCR assays for members of the Anopheles gambiae complex and Anopheles funestus group. Results: One hundred and fifty mosquitoes belonging to 11 morphological species were processed. Three species (Anopheles pretoriensis, Anopheles rufipes and Anopheles rhodesiensis) amplified members of the An. funestus group and four species (An. pretoriensis, An. rufipes, Anopheles listeri and Anopheles squamosus) amplified members of the An. gambiae complex. Conclusions: Morphological identification of mosquitoes prior to PCR assays not only saves time and money in the laboratory, but also ensures that data received by malaria vector control programmes are useful for targeting the major vectors.
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    Blood pressure measurements in the ankle are not equivalent to blood pressure measurements in the arm
    (2014-12) Goldstein, L.N.; Wells, M.; Sliwa, K.
    Background. Blood pressure (BP) is often measured on the ankle in the emergency department (ED), but this has never been shown to be an acceptable alternative to measurements performed on the arm. Objective. To establish whether the differences between arm and ankle non-invasive BP measurements were clinically relevant (i.e. a difference of ≥10 mmHg). Methods. This was a prospective cross-sectional study in an urban ED making use of a convenience sample of 201 patients (18 - 50 years of age) who were not in need of emergency medical treatment. BP was measured in the supine position on both arms and ankles with the correct size cuff according to the manufacturer’s guidelines. The arm and ankle BP measurements were compared. Results. There was a clinically and statistically significant difference between arm and ankle systolic BP (SBP) and mean arterial pressure (MAP) (–13 mmHg, 95% confidence interval (CI) –28 - 1 mmHg and –5 mmHg, 95% CI –13 - 4 mmHg, respectively), with less difference in diastolic BP (DBP) (2 mmHg, 95% CI –7 - 10 mmHg). Only 37% of SBP measurements and 83% of MAP measurements were within an error range of 10 mmHg, while 95% of DBP measurements agreed within 10 mmHg. While the average differences (or the bias) were generally not large, large variations in individual patients (indicating poor precision) made the prediction of arm BP from ankle measurements unreliable. Conclusion. Ankle BP cannot be used as a substitute for arm BP in the ED.
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    National sentinel site surveillance for antimicrobial resistance in Klebsiella pneumoniae isolates in South Africa, 2010-2012
    (South African Medical Association, 2014-08) Perovic, O.; Singh- Moodley, A.; Duse, A.; Elliott, G.; Bamford, C.; Swe Swe-Han, K.; R Kularatne, R.; Lowman, W.; Whitelaw, A.; Nana, T.; Wadula, J.; Lekalakala, R.; Saif, A.; Marais, E.
    Background: The increasing rates of antimicrobial resistance observed in the nosocomial pathogen Klebsiella pneumoniae are of major public health concern worldwide. Objectives: To describe the antibiotic susceptibility profiles of K. pneumoniae isolates from bacteraemic patients submitted by sentinel laboratories in five regions of South Africa from mid-2010 to mid-2012. Molecular methods were used to detect the most commonly found extended-spectrum beta lactamase (ESBL) and carbapenemase resistance genes. Methods: Thirteen academic centres serving the public healthcare sector in Gauteng, KwaZulu-Natal, Free State, Limpopo and Western Cape provinces submitted K. pneumoniae isolates from patients with bloodstream infections. Vitek 2 and MicroScan instruments were used for organism identification and susceptibility testing. Multiplex polymerase chain reactions (PCRs) were used to detect blaCTX-M, blaSHV and blaTEM genes in a proportion of the ESBL isolates. All isolates exhibiting reduced susceptibility to carbapenems were PCR tested for blaKPC and blaNDM-1 resistance genes. Results: Overall, 68.3% of the 2 774 isolates were ESBL-positive, showing resistance to cefotaxime, ceftazidime and cefepime. Furthermore, 46.5% of all isolates were resistant to ciprofloxacin and 33.1% to piperacillin-tazobactam. The major ESBL genes were abundantly present in the sample analysed. Most isolates (95.5%) were susceptible to the carbapenems tested, and no isolates were positive for blaKPC or blaNDM-1. There was a trend towards a decrease in susceptibility to most antibiotics. Conclusion: The high proportion of ESBL-producing K. pneumoniae isolates observed, and the prevalence of ESBL genes, are of great concern. Our findings represent a baseline for further surveillance in SA, and can be used for policy and treatment decisions.
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    Laboratory information system data demonstrate successful implementation of the prevention of the mother- child transmission programme in South Africa
    (2014-03) Sherman, G.G.; Lilian, R.R.; Bhardwaj, S.
    Background: Monitoring the prevention of mother-to-child transmission (PMTCT) programme to identify gaps for early intervention is essential as South Africa progresses from prevention to elimination of HIV infection in children. Early infant diagnosis (EID) by an HIV polymerase chain reaction (PCR) test is recommended at 6 weeks of age for all HIV-exposed infants. The National Health Laboratory Service (NHLS) performs the PCR tests for the public health sector and stores test data in a corporate data warehouse (CDW). Objectives: To demonstrate the utility of laboratory data for monitoring trends in EID coverage and early vertical transmission rates and to describe the scale-up of the EID component of the PMTCT programme. Methods. HIV PCR test data from 2003 to 2012 inclusive were extracted from the NHLS CDW by year, province, age of infant tested and test result and used to calculate EID coverage and early vertical transmission rates to provincial level. Results: Rapid scale-up of EID over the first decade of the PMTCT programme was evident from the 100-fold increase in PCR tests to 350 000 by 2012. In 2012, 73% of the estimated 270 000 HIV-exposed infants requiring an early PCR were tested and the early vertical transmission rate had fallen to 2.4% as a result of successful implementation of the PMTCT programme. Conclusions: Laboratory data can provide real time, affordable monitoring of aspects of the PMTCT programme and assist in achieving virtual elimination of paediatric HIV infection in South Africa.
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    Critical value reporting : a survey of 36 clinical laboratories in South Africa
    (2014-01) Schapkaitz, E.; Mafika, Z.
    Objective: Critical value policies are used by clinical laboratories to decide when to notify caregivers of life-threatening results. Despite their widespread use, critical value policies have not been published locally. A survey was designed to determine critical value policies for haematology tests in South Africa. Methods: A survey was carried out on 136 identified laboratories across South Africa in January 2013. Of these, 36 responded. Data collected included critical value policies, critical values for haematology parameters, and critical value reporting. Results: Of the 36 laboratories surveyed, 11.1% (n=4) were private, 33.3% (n=12) were affiliated to academic institutions and 55.6% (n=20) were peripheral or regional National Health Laboratory Service laboratories. All the laboratories confirmed that they had a critical value policy, and 83.3% of such policies were derived from local clinical opinion. Mean low and high critical limits for the most frequently listed tests were as follows: haemoglobin <6 and >20 g/dl, platelet count <41 and >1 000 ×109 /l, white cell count <2 and >46×109 /l, activated partial thromboplastin time >101 seconds, and international normalised ratio >6. In almost all cases critical value reporting was performed by the technologist on duty (97.2%). The majority of laboratories required that the person notified of the critical value be the doctor who ordered the test or the caregiver directly involved in the patient’s care (83.3%); 73.3% of laboratories indicated that they followed an algorithm if the doctor/caregiver could not be reached. Conclusion: Each laboratory is responsible for establishing clinically relevant critical limits. Clinicians should be involved in developing the laboratory’s critical value policy. The findings of this survey may be of value to local laboratories that are in the process of establishing or reviewing critical value policies