Parasitology

Scope of the Parasitology Reference Unit



  1. Provide a reference diagnostic service to NHLS and private laboratories for the human parasitic diseases.
  2. Coordinate national and international external quality assessment (EQA) programmes as follows:
    1. Provide 2 national EQA programmes for stool parasites, and blood parasites
    2. Provide a malaria EQA programme for WHO AFRO
    3. Provide a malaria EQA programme for GlaxoSmithKline Biologicals
    4. Provide a national Pneumocystis IFA EQA programme
  3. Conduct teaching and training of pathology registrars, medical scientists and technologists.
  4. Undertake national surveillance for Pneumocystis pneumonia as part of the Group for Enteric, Respiratory, and Meningeal disease Surveillance of South Africa (GERMS-SA) network.
  5. Conduct applied research in the field of human medical parasitology.

Contact Us

Staff

Collaborations

Tests

Research

Publications

External Quality Assessments

Events

2007 Annual Report

Contact us

Please direct any queries via one of the following:


e-mail:

or

e-mail:
Acanthamoeba
Acanthamoeba trophozoites stained with Giemsa

Acanthamoeba
Pneumocystis cysts stained with modified toluidine blue O
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Staff

Staff members


Pathologist-in-charge: A/Prof John Frean, MB BCh, MMed, MSc, DTM&H, FACTM
Laboratory manager: Leigh Dini, BSc (Hons), MSc
Senior medical technologist: Rita van Deventer, Dip Med Tech (Parasitol)
Medical scientist: Bhavani Poonsamy, BSc (Hons)
Medical scientist: Desiree du Plessis, BSc (Hons), MSc
Medical scientist: Benjamin Mogoye, BSc (Hons)

Data clerk:

Professional nurse:


Postgraduate students


Postdoctoral fellow:

Gayathri Venkatesan (2007-8). Malaria vector-parasite relationships.
PhD:

Leigh Dini (2005-present): A study of Pneumocystis pneumonia in South Africa.

MSc:

Kesenthri Kistiah (2007-present): Epidemiology of toxoplasmosis in South Africa.
Bhavani Poonsamy (2008-present): Resolution of mixed dihydropteroate synthase (DHPS) genotypes in respiratory specimens from patients with Pneumocystis jirovecii pneumonia from Gauteng, South Africa

BSc Honours:

V Ntigwa (2007)

John Frean


Leigh Dini


Rita van Deventrr


Bhavani Poonsamy


Desiree du Plessis


Benjamin Mogoye


Kesenthri extracting DNA for toxoplasma PCR


Gayathri culturing malaria parasites

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Collaborations
  • Chris Hani Baragwanath Hospital (Drs M Wong, A Karstaedt, A Mochan): Pneumocystis pneumonia and toxoplasmosis studies.
  • Swedish Institute for Infectious Disease Control (Drs V Fernandez, A Barragan): Pneumocystis pneumonia and toxoplasmosis studies.
  • Regional advisory group for WHO malaria EQA programme
  • Updating of WHO basic malaria microscopy manual, bench aids, slide bank SOPs and Malaria QA manual.
  • Research Institute for Tropical Medicine, Philippines (Ms J Luchavez): quality control of malaria microscopy and RDT testing, referee for GSK Malaria EQA programme
  • Department of Malaria Eradication, Oman, Dr MS Al-Zedjali: referee for GSK Malaria EQA programme
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Tests

List of reference tests we offer

(please see the Diagnostic Services section of the NICD website for a full list of parasitology tests with specimen type required and turn around times)
  • Identification of human blood parasites
  • Malaria species identification and quantitation
  • Plasmodium falciparum antigen detection
  • Wuchereria bancrofti antigen detection
  • Identification of human stool and urine parasites
  • Detection of Microsporidia
  • Worm identification
  • Immunofluorescent antigen test for Pneumocystis jirovecii
  • Detection of hydatid hooklets
  • Acanthamoeba culture

Pneumocystis pneumonia surveillance


Pneumocystis surveillance was in launched May 2006 using the current GERMS-SA enhanced sites. The aims are to assess the burden of Pneumocystis pneumonia (PcP) in South Africa, to investigate co-trimoxazole resistance, and to train and strengthen diagnostic capacity amongst laboratories.
Course participants and registrars receiving training by PRU staff












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Research

Current Research Projects


Pneumocystis jirovecii project
PRU, in collaboration with the Swedish Institute for Infectious Disease Control, is conducting a research study to survey drug-resistant strains of Pneumocystis jirovecii in South Africa. Quantitative real-time PCR has been set up in the unit and staff members received training in 2006 and 2007. A related project, ‘Utility of S-adenosylmethionine as a plasma marker of infection with P. jirovecii' has received MRC funding.

Toxplasmosis project
A study of the epidemiology of toxoplasmosis in South Africa, in collaboration with The Swedish Institute for Infectious Disease Control, was launched in 2007. We are investigating the seroprevalence of toxoplasmosis in Johannesburg among healthy individuals and populations at risk for severe complications, ie HIV-positive individuals and pregnant women. Furthermore, we will isolate and genotype local Toxoplasma strains from humans and rodents to identify and define molecular virulence markers relevant for severe toxoplasmosis. The results will have direct implications for establishing strategies to combat and prevent this potentially lethal opportunistic disease.

In vitro malaria gametocyte production
As part of a collaborative research project with the Vector Control Reference Unit of the NICD, the PRU established in vitro cultures of a laboratory-adapted Plasmodium falciparum strain. While cultures have been successfully maintained for many months, it has proved very difficult to reliably infect vector mosquitoes with malaria parasites and these techniques are being refined.

Malaria PCR
Malaria species-specific PCR is being set up to verify all EQA specimens.
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Publications
  • Frean J, Blumberg L, Cutland C. Q fever: the southern African perspective. Ann Aust Coll Trop Med 2007; 8: 32-34.
  • Blumberg L, Weyer J, Frean J, Ogunbanjo GA. Rabies: an evidence-based approach to management. S Afr Fam Practice 2007; 49: 35-41.
  • Blumberg L, Frean J. Malaria control in South Africa: challenges and successes. S Afr Med J 2007; 97: 1193-1197.
  • Frean J, Blumberg L. Tick bite fever and Q fever – a South African perspective. S Afr Med J 2007; 97: 1198-1202.
  • Frean J. Microscopic images transmitted by mobile cameraphone. Trans Roy Soc Trop Med Hyg 2007; 101: 1053.
  • Frean J. Bilharzia. In: Snyman J (ed). MIMS Primary Care, Vol. 1. MIMS, Johannesburg, 2007, 143-146.
  • Goldsmid J, Frean J. Parasitic Diseases. In: Kibel MA, Saloojee H (eds). Child Health for All, 4th Edition. Oxford University Press, Cape Town, 2007.
  • Dini L, Wong ML, du Plessis M, Karstaedt A, Fernandez V, Frean J. 2006. Prevalence of DHPS polymorphisms associated with sulfa resistance in South African Pneumocystis jirovecii strains. J. Eukaryot. Microbiol.; 53 (Sl):S110-1.
  • Leggatt PA, Frean J. Health countermeasures for military deployment (addendum). Occupational Health Southern Africa (2006); 12: 4-12.
  • Frean J, Blumberg L. Major rickettsial diseases in South Africa. In: Snyman JR (ed). (2006). MIMS Disease Review. MIMS, Johannesburg.
  • Blumberg L, Frean J, Baker L. (2006) Clinical Management of Malaria. Foundation for Professional Development, Pretoria.
  • Dini L, du Plessis M, Karstaedt A, Fernandez V, Frean J. (2006). Prevalence of DHPS polymorphisms associated with sulfa resistance in South African Pneumocystis jirovecii strains. J. Eukaryot. Microbiol.; 53 Suppl 1:S110-1.
  • Obihara CC, Beyers N, Gie RP, Hoekstra MO, Fincham JE, Marais BJ, Lombard CJ, Dini LA, Kimpen JLL. (2006). Respiratory atopic disease, Ascaris-immunoglobulin E and tuberculin testing in urban South African children. Clinical and Experimental Allergy; 36:640-648.
  • Frean J. Parasitic infections in the ICU. In: Feldman C, Sarosi GA, (eds). (2005) Tropical and Parasitic Infections in the Intensive Care Unit. Springer, New York.
  • Blumberg L, Frean J. The returning traveller with fever. CME - The SA Journal of CPD (2005); 23: 138.
  • du Plessis M, Dini L, Klugman KP. (2005). Correlation of PCR and IFA for detection of Pneumocystis jirovecii from sputum samples. South Afr J Epidemiol Infect; 20 (3): 109.
  • Dini L. (2005). Laboratory diagnosis of P. jiroveci. South Afr J Epidemiol Infect; 20 (3): 110.
  • Dini LA, Frean JA. (2005). Clinical significance of mites in urine. Journal of Clinical Microbiology; 43 (12): 6200-6201.
  • Cohen C, Karstaedt A, Frean J, Thomas J, Govender N, Prentice E, Dini L, Galpin J, Crewe-Brown H. (2005). Increased prevalence of severe malaria in HIV-infected adults in South Africa. Clinical Infectious Diseases: 41 (11): 1631-7.
  • Dini LA, Frean JA. (2005). Proposed guidelines for malaria antigen testing. South African Medical Journal; 95 (6): 411-412.
  • Frean J, Arntzen L, Klugman K. Susceptibility of Bacillus anthracis to moxifloxacin. Southern African Journal of Epidemiology and Infection (2005); 20: 119-120.
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External Quality Assessment (proficiency testing)
PRU coordinates 4 EQA programmes and participates in 3 international EQA programmes: CAP blood parasites, CAP stool parasites and SMI Pneumocystis inter-laboratory comparison. In 2008 a Pneumocystis IFA EQA programme will be launched.


Preparation of blood films, mass staining and stock control for EQA
WHO Malaria Microscopy EQA
A malaria microscopy EQA programme, launched in September 2005, provides a quality assessment programme to 65 public health laboratories in Africa on behalf of WHO AFRO. Surveys are conducted 3 times per year with 10 microscopy challenges for species identification and parasite quantitation per survey.

GSK malaria EQA
PRU has been contracted by GlaxoSmithKline since January 2006 to provide a malaria microscopy and quantitation EQA programme for their malaria vaccine sites in Africa. Surveys are conducted 3 times per year with 20 microscopy challenges for species identification and parasite quantitation per survey.


Staff from PRU and CLS involved in the GSK malaria EQA programme


NICD Parasitology EQA Programmes
PRU coordinates 2 EQA programmes for the NHLS with over 200 participants from southern Africa: Stool parasites, and Blood parasites. The aims of the programmes are to build capacity in the field of human diagnostic parasitology in southern Africa, and to obtain an objective measure of the diagnostic ability of participants. EQA surveys are conducted 3 times a year and comprise of at least 4 stool or blood specimens, including a laboratory technique challenge. A teaching series is included in each survey to encourage participants to learn more about medically important parasites. Both Parasitology EQA programmes are CPD accredited.

Participants are assessed on their ability to accurately identify stool or blood parasites; correctly stain a blood or stool smear; perform parasite counts; and provide results on time with the correct details. Results are analysed using a system of referee laboratories to guide the standard for assessment of participants. The model (expected) results are compared against those returned by the referees. There must be 70% agreement among referees for a challenge to be scored. An individual report is issued to each participant detailing their performance and an analysis of the performance of all other unnamed participants. It is accompanied by a detailed commentary, which includes illustrations of the parasites found in that particular survey, comments on pitfalls and errors, additional educational material and a corrective action form. Regional summaries and completed corrective actions for NHLS participants are sent to the regional managers. Executive summaries are sent to the CEO, QA Division Head and NICD Director.
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Events
Upcoming events in 2008
  • Registrar training, 3-7 March and 14-18 July 2008.
  • Audit of malaria EQA programme by GlaxoSmithKline Biologicals, 5 March 2008
  • Malaria EQA course jointly organised with WHO for participants from Nigeria and Oman, 15-17 April 2008.
  • Malaria microscopy and quantitation on-site training in Burkina Faso in collaboration with GlaxoSmithKline Biologicals, 21-25 April 2008.
  • Laboratory diagnosis of stool parasites, 1-3 July 2008.
  • SANAS assessment, 15 October 2008
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2007 Annual Report: Parasitology Reference Unit (PRU), NICD

Achievements

The unit was audited by SANAS in November 2008 and achieved accreditation to ISO15189.
J. Frean was elected Fellow of the Faculty of Travel Medicine, Royal College of Physicians and Surgeons of Glasgow


Visiting guests

Drs Antonio Barragan and Victor Fernandez, Swedish Institute for Infectious Disease Control
Ms Jen Daily, Clinton Foundation HIV/AIDS Initiative
Ms Jenny Luchavez, Research Institute for Tropical Medicine, Philippines


Meetings attended and talks presented

  • Staff members regularly attended and performed presentations for the Friday journal club.
  • Staff participated in the WHO Regional Advisory Group meeting, at the NICD in Johannesburg. Malaria EQA results were presented and discussed for the Afro region.
  • Frean J. African Tick bite fever. RSTMH Centennial Symposium, 16th Annual Scientific Meeting of the Australasian College of Tropical Medicine and North Queensland Centre for Cancer Research, Townsville, Australia, July 2007.
  • Frean J. Tales of the unexpected: malaria acquired outside endemic areas. 2nd Copeland oration, 16th Annual Scientific Meeting of the Australasian College of Tropical Medicine and North Queensland Centre for Cancer Research, Townsville, Australia, July 2007.
  • Blumberg L, Frean J. Malaria in the concrete jungle. 36th Annual Conference of the Parasitological Society of Southern Africa, Malelane, South Africa, September 2007.
  • Dini L, Wong ML, du Plessis M, Karstaedt A, Frean J, Fernandez V. Dihydropteroate synthase polymorphisms in Pneumocystis jirovecii in South Africa. Presentation at Molecular and Cell Biology Symposium, Pretoria, 2007.
  • Dini L, Wong ML, du Plessis M, Karstaedt A, Frean J, Fernandez V. Pneumocystis pneumonia and markers of resistance. Presentation at GERMS-SA Principal Investigator Meeting, Johannesburg, 2007.
  • Wong ML, Dini L, Frean J, du Plessis M, Fernandez V, Karstaedt A. 2007. Dihydropteroate synthase (DHPS) polymorphisms in patients with Pneumocystis jirovecii pneumonia at Chris Hani Baragwanath Hospital. Poster presentation at the South African Thoracic Society Congress, Johannesburg, South Africa, 2007.
  • Dini L, du Plessis M, Beser J, Wong M, Karstaedt A, Frean J, Fernandez V. Frequency of DHPS polymorphisms associated with sulfa resistance in South African Pneumocystis jirovecii strains. Poster presentation at 2nd FEBS Advanced Lecture Course Human Fungal Pathogens, Nice, France, 2007.

Training provided by the Unit

Trained 16 participants over 2 days in the laboratory diagnosis of malaria through theory and practical sessions.
7 registrars received training in parasitology
Trained J. Luchavez, from the Research Institute for Tropical Medicine (RITM) in the Philippines, in the preparation and mass staining of blood films.
R. van Deventer set up parasitology practicals at Wits University for 200 1st year medical students and 30 3rd year BSc students.



Participants and lecturers of the “Malaria course



Jenny Luchavez from RITM in the Philippines receiving training in blood film preparation



Training that staff received

L. Dini was trained in molecular biology (robotic DNA extraction, quantitative real-time PCR, molecular genotyping, cloning) for 4 months at the Swedish Institute for Infectious Disease Control
B. Poonsamy attended a SANAS laboratory accreditation course and a GLP course
B. Poonsamy and R. van Deventer attended safety courses
B. Mogoye and R. van Deventer attended basic fire training
L. Dini and B. Poonsamy attended an “Excellence in reporting science workshop” by Dr E. Churchill from CDC Atlanta Georgia, USA.
Staff received training in robotic DNA extraction using the Roche MagNa Pure Compact.



L. Dini and staff from the Swedish Institute for Infectious Disease Control



Pneumocystis pneumonia surveillance

The total number of cases acquired by the GERMS-SA surveillance system in 2007 was 286, of which 252 were laboratory-confirmed. Compared with other opportunistic pathogens under surveillance, this is certainly a substantial underestimate of the burden of disease. There are several reasons for this: the condition is mainly clinically diagnosed and laboratory diagnosis is only offered in a few larger centres.

Quality assessment activities

WHO Malaria Microscopy EQA
Three surveys consisting of 10 microscopy and quantitation challenges were sent to 65 public health laboratories in Africa.

GSK malaria EQA
Three surveys consisting of 20 microscopy and quantitation challenges were sent to 9 malaria vaccine sites in Africa. The challenges were carefully planned to assess participants’ ability to identify malaria species and accurately quantify parasites. The consistency of a participant’s parasite counting ability was assessed by inserting duplicate specimens into surveys. Training in parasite quantitation was conducted at a site in Tanzania.

NICD Parasitology EQA Programmes
Please see annual report below

2007 Annual Report for Parasitology NICD EQA Programmes


Stool EQA Programme for 2007: Survey contents and results



Table 1: 2007 Survey Challenges- Stool EQA Programme
Specimen code Challenge type Description of challenge

Survey 1 for 2007
PS1/07 Microscopy and ID Stool concentrate with Hymenolepis nana ova
PS2/07 Microscopy and ID Stool concentrate with no parasites present
PS3/07 Modified ZN stain, microscopy and ID Stool smear with Cryptosporidium species oocysts
PS4/07 Modified ZN stain, microscopy and ID Stool smear with Cryptosporidium species oocysts
Teaching booklet Bench aids and SOPs

Survey 2 for 2007
PS507 Microscopy and ID Stool concentrate with Giardia lamblia cysts
PS6/07 Microscopy and ID Stool concentrate with Ascaris lumbricoides & Trichuris trichiura
PS7/07 Modified ZN stain, microscopy and ID Stool smear with no parasites present
PS8/07 Modified ZN stain, microscopy and ID Stool smear with Isospora belli oocysts
Teaching series Microscopy tips and stool artefacts

Survey 3 for 2007
PS9/07 Microscopy and ID Stool concentrate with Strongyloides stercoralis larvae
PS10/07 Microscopy and ID Stool concentrate with hookworm ova & Entamoeba coli cysts
PS11/07 Microscopy and ID Stool concentrate with no parasites present
PS12/07 Smear preparation, modified ZN stain, microscopy and ID Stool concentrate with no parasites present
Teaching series Life cycle of Hymenolepis nana


Table 2: 2007 Results- Stool EQA Programme
  Survey 1 Survey 2 Survey 3 2007 Total
Number of participants 217 218 218 218
Number of non-returns 23 19 25 5
Average score 58.1% 60.7% 59.3% 59.3%
Number of participants with scores of 100% 61 60 56 22
Number of participants with scores of =50% 133 138 142 137
Number of participants with scores of <50% * 84 80 76 81

* Includes non-returns who automatically receive 0


Figure 1: 2007 Results- Stool EQA Programme




Blood EQA Programme for 2007: Survey contents and results




Table 3: 2007 Survey Challenges- Blood EQA Programme
Specimen code Challenge type Description of challenge

Survey 1 for 2007
PB1/07 Microscopy & ID Unstained thick & thin blood films with no parasites present
PB1a/07 Assessment of stain quality Unstained thick blood film with no parasites present
PB1b/07 Assessment of stain quality Unstained thin blood film with no parasites present
PB2a & b/07 Microscopy & ID Giemsa stained thick & thin blood films with P. falciparum
Teaching booklet Bench aids & SOPs

Survey 2 for 2007
PB3 & 4/07 Microscopy & ID Giemsa stained thick & thin blood films with Trypanosoma brucei species
PB5 & 6/07 Microscopy & ID Giemsa stained thick & thin blood films with P. falciparum
PB5/07 Parasite count (thick) Giemsa stained thick blood film with P. falciparum
PB6/07 Parasite count (thin) Giemsa stained thin blood film with P. falciparum
Teaching series Malaria parasite counting methods

Survey 3 for 2007
PB7/07 Paper challenge ID Loa loa microfilariae
PB8 & 9/07 Microscopy & ID Giemsa stained thick & thin blood films with P. falciparum
PB8/07 Parasite count (thick) Giemsa stained thick blood film with P. falciparum
PB9/07 Parasite count (thin) Giemsa stained thin blood film with P. falciparum
Teaching series Components of blood


Table 4: 2007 results - Blood EQA Programme

  Survey 1 Survey 2 Survey 3 2007 Total
Number of participants 196 197 198 198
Number of non-returns 20 19 17 8
Average score 47.6% 80.2% 55.2% 60.7%
Number of participants with scores of 100% 11 98 18 1
Number of participants with scores of =50% 123 183 134 148
Number of participants with scores of <50% * 73 14 64 50

* Includes non-returns who automatically receive 0


Figure 2: 2007 Results- Blood EQA Programme




Analysis and general comments

As with previous years, there are two issues of concern: the high rate of non-returns for each survey and the number of laboratories failing to meet an acceptable standard. The standard of these two NICD Parasitology EQA Programmes is set at a minimum level for a routine diagnostic laboratory, so participants should easily achieve over 50%. However 37% of participants for the Stool EQA Programme failed and 25% failed the Blood EQA Programme. Participants that fail the Parasitology EQA Programmes should preferably not offer parasitic diagnosis until they are able to sustain a pass grade.

True values

However, as the scoring system is not linear (see table below), the actual criterion for acceptable results is an average total score of =75%, rather than 50%. Therefore, only 36% and 31% of participants reached the target score for the Stool Programme and the Blood Programme respectively.

Figure 3: Acceptable versus unacceptable results for the 2007 Stool EQA Programme



Figure 4: Acceptable versus unacceptable results for the 2007 Blood EQA Programme



Table: Grading system for NICD Parasitology EQA Programmes
Score: Result: Definition: Performance assessment:
4 Completely correct result A result accepted as the most correct and clinically relevant result. Acceptable
3 Almost completely correct result A result not technically correct, but the error or omission has little or no clinical impact; a deviation from what is considered the most clinically relevant result. Acceptable
Separator To divide the acceptable from unacceptable responses. n/a
1 A significantly incorrect result A clinically relevant result that could lead to a minor diagnostic or treatment error. Unacceptable
0 Completely incorrect result A clinically relevant result that could lead to a major diagnostic or treatment error. Unacceptable
0 No result No result submitted by participant. Unacceptable


Performance over the last 4 years
The following graphs illustrate participant performance over the last 4 years. There was a decrease in the percentage of laboratories that passed in each programme from 2006 to 2007. This could be due to the large increase of participants in these programmes.


Figure 5: Comparisons of participant performance in the Stool EQA Programme, 2004-2007



Figure 6: Comparisons of participant performance in the Blood EQA Programme, 2004-2007
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