Annual Report March 2006 – March 2007
Respiratory & Meningeal & Pathogens Research Unit
Departmental Staff Compliment
Directors: Professor Keith P Klugman/ Assoc. Prof. Shabir A Madhi
Staffing at NICD: One Senior Pathologist, 1 Senior Scientist, 1 Laboratory Manager, 3 Scientists, 2 Senior Medical Technologists, 1Medical Technologist, 1 Junior Technologist, 1 Data Clerk, and 1 Personal Assistant to the Director.
Staffing at ChrisHaniBaragwanathHospital: Co-Director: Associate Professor Shabir Madhi.
One Senior Medical Officer, 11 Research Medical Officers, 1 Senior Scientist, 1 Physician, 1 Statistician, 1 Scientist, 1 Study Co-coordinator, 19 Professional nurses, 4 Enrolled nurses, , 5 Nursing Assistants, 1 Nurse, 5 Laboratory Technologists, 1 Clinical Trial co-coordinator, 6 Field Workers, 9 Research assistants, 4 Counselors, 3 Data clerks, 3 Administrators, 2 Drivers, 1 Cleaner.
Departmental Research Interests
Acute Respiratory infections are the leading cause of death in children in developing countries and the leading infectious cause of death in all ages. The Respiratory and Meningeal Pathogens Research Unit (RMPRU) is mandated to study the causes of respiratory and meningeal infections. Both clinical and basic research is conducted with the aim of improving the diagnosis, management and prevention of these diseases.
The structure of the Unit has changed to accommodate the move of the surveillance arm of the Unit to NICD. The Unit’s functions are broadly sub-divided into epidemiological and basic science research (with a focus on antimicrobial resistance) and clinical research largely focusing on vaccinology. The aspect of the research unit affiliated to Wits is based at Chris Hani Baragwanath hospital under the supervision of Prof Madhi while the surveillance arm is at NICD at Rietfontein under the supervision of Dr. von Gottberg. Prof Klugman retains overall responsibility for the MRC Unit at both sites and Prof Madhi has been appointed as a co-director of the Unit. The division of the unit based at CHB has a total staff complement of 95, whom with the exception of one posted funded through MRC funds, are funded exclusively through grant-funds.
The research currently underway at the clinical/vaccinology division of the Unit based at CHB hospital include studies on: 1. the molecular and clinical epidemiology of newly discovered respiratory viruses such as echovirus, coronavirus, bocavirus and rhinovirus. Additionally, the role of pneumococcal coinfections in severe episodes of such viral infections is being probed; 2. The effect of HIV exposure and treatment with anti retroviral drugs on the quantitative and qualitative antibody responses to conjugate bacterial vaccines aimed at preventing respiratory tract infections; 3. affordable and implemental interventions in reducing neonatal sepsis, a large proportion of which is attributed to respiratory disease due to S. agalactiae, is underway; 4. the epidemiology of pneumococcal pneumonia in adults and the role of newer diagnostic modalities in improving on making a pathogen specific diagnosis in adults with suspected pneumococcal pneumonia; 5. prevention of tuberculosis in HIV infected children by way of primary prophylaxis with Isoniazid between 3 and 24 months of age; 6. The molecular and clinical epidemiology of Staph aureus infections; 7. Defining the correlates of protection against nasopharyngeal colonization by pneumococci and the mechanism of protection induced by conjugate pneumococcal vaccines.
During 2006 Profs Madhi and Klugman completed the chapter on Acute Respiratory Infections for the World Bank book on Disease and Mortality in Sub-Saharan Africa. Research from the Unit has led to the proposition of a new algorithm to measure the burden of pneumococcal disease preventable by conjugate vaccine. The Unit’s expanded national surveillance for respiratory pathogens continues to be supported by CDC/USAID. We documented and published in the Bulletin of WHO the impact of Haemophilus influenzae type b (Hib) vaccine on invasive disease in South Africa. The burden of invasive Hib has been greatly reduced by the introduction of conjugate Hib vaccine, despite the impact of the HIV epidemic which has increased invasive Haemophilus influenzae disease burden due to serotypes other than type b. The Unit has demonstrated for the first time the clinical and molecular epidemiology of human metapneumovirus infection in children in a developing country. During 2006 studies the Unit was successful in attracting funding through the Gates Foundation Grand Challenges. This has allowed us to extend our study funded by NIH, USA on the immunological responses to pneumococcal conjugate vaccine in HIV infected children, and we have enrolled the majority of adults in an NIH funded study to measure the burden of vaccine–preventable disease in HIV–infected adults. Another major ongoing study in the Unit is the recruitment of infants at birth to evaluate the impact of chlorhexidine washes for the mother and infant on neonatal sepsis.
Research Projects
Human metapneumovirus pneumonia in HIV-infected and -uninfected children
Project leader: Prof S Madhi. Researcher: Mr Herbert Ludewick. Collaborator: Professor Guy Boivin, University of Quebec, Canada.
In addition to the study published on the molecular epidemiology of human metapneumovirus over three consecutive epidemics in South Africa in Emerging Infectious Diseases in 2005, the first of its kind from any developing country, we have now also published on the role of pneumococcus in the pathogenesis of severe hMPV infections in Journal of Infectious Diseases. Additionally, differences in the clinical characteristics between HIV infected and HIV uninfected children have been described and are currently in press.
Risk factors and clinical course of pneumococcal invasive disease in HIV infected and uninfected adults and children
Project leaders: Professor Charles Feldman, Prof Shabir Madhi. Collaborators: Dr Victor Yu, University of Pittsburgh, Dr Leslie McGee, EmoryUniversity, Atlanta.
Funded by the MRC/NHLS/Wits.
This definitive study was conducted in collaboration with Dr Victor Yu (University of Cleveland). The Unit contributed cases of pneumonia, and adults with meningitis in collaboration with Prof C Feldman. The study from 2002 included pneumococcal meningitis in children. The initial study on bacteraemic pneumonia was published in 2003 in Clinical Infectious Diseases. Further analyses of the HIV - infected population included in the study have been completed. An analysis of combination therapy showed that this was associated with decreased mortality in severely ill patients and was published in 2004. The impact of serotypes on mortality has been submitted during 2006 to Clinical Infectious Diseases and the analysis of the molecular relatedness of the strains has been completed
Impact of Hib conjugates vaccine on the burden of invasive Haemophilus influenzae type b.
Project leaders: Dr A von Gottberg. Collaborators: Dr A Schuchat, Dr S Schrag, CDC, Atlanta, USA. Funding MRC/NHLS/Wits, USAID/CDC/PEPFAR.
Following our initial studies on the long term level of protection afforded by Hib vaccine in HIV- infected children, a national study on the effect of HibCV on the burden of invasive Hib disease in South Africa was published in the Bulletin of the WHO in 2006.
Analysis of the genetic diversity of Neisseria meningitidesin South Africa
Project leaders: Dr Anne von Gottberg, Mr Garry Coulson, Mignon du Plessis. Funded by the MRC/NHLS/WITS.
A molecular epidemiology study of Neisseria meningitides (the meningococcus) in South Africa, for the period July 1999 to July 2002 has been completed. Over 600 isolates have been characterized by pulsed-field gel electrophoresis and over 40 representative isolates of interest were further characterized by multi-locus sequence typing. The results of this study indicate that for each of the five serogroups studied (A, B, C, W135 and Y), hypervirulent clones/clonal complexes, known to have repeatedly caused epidemics and outbreaks worldwide, are circulating in the South African population. The continuous monitoring of the changing epidemiology in South Africa of the clonal composition of meningococci in South Africa has direct implications for public health decisions, with regard to the development of effective control and intervention strategies. These data will be published in Emerging Infectious Diseases Journal early in 2007.
Telithromycin resistance mechanisms in the pneumococcus
Project leader: Nicole Wolter. Funded by the MRC/NHLS/WITS.
Macrolides are commonly used for the treatment of pneumococcal infection. However, due to the global increase in macrolide resistance alternatives are being developed. Telithromycin, a semi-synthetic derivative of erythromycin, is the first ketolide to be approved for clinical use. The structural modifications of this antibiotic enable it to form a stronger bond with the ribosome than erythromycin and it can therefore overcome the common macrolide resistance mechanisms, erm (B) and mef (A). Although pneumococcal resistance to telithromycin remains rare, resistant isolates are emerging. The mechanism of resistance in a rare highly telithromycin-resistant isolate was investigated. The isolate was found to containerm (B) with a truncated leader peptide and a mutant ribosomal protein L4. By transformation of susceptible strains, this study showed that high-level telithromycin resistance is conferred by erm (B), wild-type or mutant, in combination with a 69GTG71 to TPS mutation in ribosomal protein L4. This work has been submitted to Antimicrobial Agents and Chemotherapy for publication. A telithromycin-resistant isolate with a 136 bp deletion in arm (B) is currently being investigated for the mechanism of telithromycin resistance.
Macrolide resistance in Streptococcus pneumoniae causing invasive disease in South Africa: 2000 – 2005
Project leader: Nicole Wolter. Funded by the MRC/NHLS/WITS.
Resistance to macrolides in Streptococcus pneumoniae is conferred by two predominant mechanisms namely, active drug efflux and target-site modification. Drug efflux occurs due to acquisition of the me (A) gene which confers resistance to 14- and 15-membered macrolides (M-phenotype). Target modification occurs predominantly due to acquisition of erm (B). This gene encodes a ribosomal methylase that methylates A2058of 23S rRNA, conferring cross-resistance to the macrolide-lincosamide-streptogramin B antibiotics (MLSB phenotype). Target modification may also arise from mutations in the genes encoding domain V of 23S rRNA and the ribosomal proteins L4 and L22. There is an increase in macrolide resistance globally, and the predominant mechanism of resistance varies geographically. In this study, macrolide resistance in invasive pneumococcal isolates collected in South Africa for the period 2000-2005 will be investigated. Preliminary data show an increase in macrolide resistance from 9% in 2000 to 14% in 2005. The predominant phenotype is the MLSB phenotype (75%). Serotype 14 is most prevalent in macrolide-resistant strains. Random isolates from 2005 will be genotypically screened for the presence of erm(B) and mef(A) genes.
Molecular epidemiology of South African serotype 3 pneumococci
Project leaders: Anne von Gottberg, Kedibone Mothibeli, Mignon du Plessis. Funded by MRC/NHLS/WITS.
Pneumococcal serotype 3 is an important cause of invasive disease worldwide. Patients infected with this serotype have also been shown to have higher fatality rates compared to those infected with other invasive serotypes such as 1 and 14. The aim of this study was to investigate the genetic clonality of South African pneumococcal serotype 3 isolates. For this purpose, a total of 102 serotype 3 isolates were selected from isolates recovered from specimens of patients of all ages from Gauteng province. All specimens were collected from January 2000 to December 2002. These 102 isolates from blood and/or CSF have been analyzed by pulsed-field gel electrophoresis (PFGE). A total of 18 isolates selected from each of the PFGE clusters were further analyzed by multi locus sequence typing (MLST). The results suggest that South African serotype 3 isolates are clonally diverse, and that the predominant clone circulating among the South African serotype 3 isolates differs from the predominant global clone of serotype 3.
Molecular epidemiology of multidrug-resistant pneumococcal serotype 19A isolates from South Africa
Project leaders: Kedibone Mothibeli, Mignon du Plessis, Anne von Gottberg, Funded by the MRC/NHLS/WITS.
In 1977, the first penicillin- and multidrug-resistant serotype 19A strain was isolated from a patient in South Africa. Serotype 19A continues to be prevalent in South Africa in young children and is often associated with multidrug resistance. To determine if this 1977 clone is still circulating among recent multidrug-resistant serotype 19A strains, we compared multidrug-resistant serotype 19A isolates collected from June 1999 to December 2004 in South Africa with this 1977 strain. During the study period 14 177 cases of IPD were reported. Of these, 13 064 (92%) had viable isolates available for further testing and 885 (7%) were identified as serotype 19A. Reduced susceptibility to penicillin was detected in 235 (27%) of these isolates, 25 (11%) of which were multidrug-resistant. PFGE was performed on all multidrug-resistant strains. PFGE analysis demonstrated diversity among these isolates. MLST of 15 randomly selected isolates revealed several sequence types (ST), none of which were the same as the 1977 clone.
Penicillin non-susceptibility in meningococcal isolates causing invasive disease in South Africa
Project leaders: Mignon du Plessis, Anne von Gottberg, Linda de Gouveia. Funded by MRC/NHLS/Wits.
Neisseria meningitidis non-susceptible to penicillin (MIC ≥0.12g/ml) has been reported in several parts of the world, but to our knowledge not from the African continent. In some countries, non-susceptible invasive isolates have been associated with a single clone. Data were analyzed from January 2000 through December 2005 from a national laboratory-based surveillance program in South Africa. 2130 cases of invasive meningococcal disease were reported during the 6 years analyzed, with an average annual incidence rate of 0.77/100 000 population. Of these cases, 1579 (74%) had viable isolates available for further testing, of which 97 (6%) tested non-susceptible to penicillin. Prevalence rates fluctuated over the years and genotyping showed that penicillin-resistant meningococcal isolates analyzed in this study were not clonal and were represented by all serogroups distributed throughout all provinces and age groups.
Molecular characterization of serogroup B meningococcal isolates causing invasive disease in South Africa
Project leaders: Anne von Gottberg, Mignon du Plessis, Linda de Gouveia. Collaborator: Dr. Gary Zlotnick (Wyeth Pharmaceuticals). Funding: Wyeth
Currently there is no licensed vaccine for prevention of bacterial meningitis caused by serogroup B N. meningitidis. Outer membrane protein LP2086 has been shown to elicit a good antibody response in animal models and is therefore a promising vaccine candidate. The aim of this study is to investigate the prevalence, distribution and sequence diversity of LP2086 in both serogroup B and other serogroups of meningococci circulating in South Africa.
Evaluation of Real-Time PCR and a Luminex-based assay for diagnosis of pneumococcal disease
Project leaders: CDC (Respiratory Diseases Branch and Meningitis and Special Pathogens Branch). Collaborators: Mignon du Plessis, Olga Perovic (Jhb Hospital), Jeannette Wadula (Bara). Funding: CDC
The purpose of this study is to evaluate 5 Real-Time PCR assays and a Luminex-based assay for detection of S.pneumoniae using isolates and clinical specimens from different sources. Blood and CSF specimens are currently being collected from CH Baragwanath and JohannesburgHospitals and will be sent over to the CDC for testing.
Immunogenicity and functionality of antibody induced by pneumococcal conjugate vaccine in HIV- infected infants
Project leaders: CDC (Respiratory Diseases Branch and Meningitis and Special Pathogens Branch). Collaborators: Mignon du Plessis, Olga Perovic (Jhb Hospital), Jeannette Wadula (Bara). Funding: CDC
We obtained initial funding for this study from WHO. We used cohorts of children in whom we had data on immunogenicity. HIV status was determined in an unlinked anonymous way. This collection of sera was started in 2001 and completed in 2002. The analysis of the results was completed in 2004. The Unit has sought and obtained a CIPRA grant to expand this study and a large multi- year project has been designed. An antibody functionality laboratory has been set up by Dr Peter Adrian who joined the Unit again after a number of years in Europe. He attended training during 2003 with our collaborator in Finland. The first series of experiments have been completed and the data were accepted and published during 2005 in PIDJ.
Impact of cotrimoxazole prophylaxis on resistance in respiratory pathogens
Project leaders: Dr A von Gottberg, Dr V Quan, L de Gouveia. Collaborators: Dr Anne Schuchat, CDC, Allison Taylor, EmoryUniversity. Funding: CDC-USAID
The widespread introduction of cotrimoxazole for prophylaxis of opportunistic infections in HIV- infected people has major implications for the development of resistance and the selection of multiresistant bacterial clones. We have, with CDC and EmoryUniversity, expanded our national surveillance network to monitor the impact on resistance and the effectiveness of the intervention on pneumococcal disease. Funding has been secured from USAID via the CDC. Enhanced surveillance commenced in 2003. Currently there are 10 sites involved nationally. Data are disseminated to participants by quarterly reports and LINK newsletter. An analysis conducted in 2004 shows dramatic increases in cotrimoxazole resistance in pneumococcal isolates. This study will be submitted for publication in 2007. Group for Enteric-Respiratory and Meningeal Disease Surveillance in South Africa (GERMS-SA).
Differences in blood culturing practices in rural and urban areas of South Africa
Project leader: Dr Anne von Gottberg, Project coordinators: Dr Vanessa Quan and Ms Linda de Gouveia
Funding: Gates Foundation PNEUMO_ADIP.
It has recently been suggested that the burden of invasive bacterial disease in Africa is greater than was previously thought, but there are many obstacles to accurately measuring this disease burden. In South Africa there are good data on invasive pneumococcal disease for some urban centres, but limited information of disease burden exists outside of these areas.
We proposed to improve the quality of surveillance data in South Africa, by collecting details of laboratory and clinical blood culturing practices throughout the country. We will investigate the quality of laboratory processing of specimens. Twenty-one hospitals throughout the country have been approached to request doctors working in these hospitals to fill in a questionnaire with regard to microbiology laboratory diagnostics, specifically blood cultures, pneumococcal disease burden and the availability of possible intervention strategies.
These data will assist in the interpretation of ongoing surveillance for invasive pneumococcal disease. We also hope to strengthen laboratory networks in South Africa. Funding for the proposal has been received from the Gates Foundation Pneumo-ADIP.
Molecular Epidemiology of Haemophilus influenzae