Evaluation of MODS (Microscopic Observation Drug Susceptibility assay) for diagnosis of Tuberculosis in Ethiopia. A feasibility study.

Over 5,000 people die of tuberculosis every day. The majority of this avoidable morbidity and mortality is in the developing world. Ethiopia is one of the 22 high burden countries in the world. The DOTS strategy has had some notable, but limited, success in turning the tide of this growing burden and the HIV pandemic and the emergence multi-drug resistant disease (MDR-TB) worldwide demands urgent improvement of control efforts.

The diagnosis of TB by sputum smear microscopy is the standard method and an integral feature of the DOTS (direct observation of treatment-short-course chemotherapy) strategy for global TB control. Sputum smear diagnosis is simple, low cost and the most important diagnostic tool in the developing world. Nonetheless, the sensitivity of microscopy for the detection of all cases is low, even when the optimum sensitivity of microscopy is achieved (approximately half of all culture-positive cases are smear negative), and the performance of microscopy is highly variable. Furthermore, the contribution of transmission of infection by smear-negative culture-positive patients is not inconsiderable, and the potential impact of the detection and treatment of these patients is significant.

In the developing world, the high prevalence of HIV/AIDS and the absence of information on resistance and susceptibility testing threaten the continuing role of the sputum smear as the sole tool for the diagnosis of the majority of cases of TB worldwide. The development of new, low-cost diagnostic tools offers the possibility of future TB control on the basis of culture-based diagnosis and more widespread, targeted susceptibility testing.

In Ethiopia, diagnosis of TB mainly relies on sputum smear microscopy. The high HIV/TB co-infection rate (40-50%) is making the diagnosis of TB by sputum smear very difficult. The number of TB treatment failure cases is increasing year by year, calling for a need to have a feasible means of drug resistance testing. Therefore, having a simple, cost effective diagnostic method in the Ethiopian context will improve the TB program and the TB/HIV collaborative activity.

Microscopic observation drug susceptibility assay (MODS) is a rapid, efficient detection and drug susceptibility testing of Mycobacterium tuberculosis in sputum by microscopic observation. It is especially fit for developing countries like Ethiopia.

This project tries to assess the feasibility of MODS and to examine the added value of MODS over the current TB diagnostic methodology in Ethiopia.

The project has three phases. The first phase, in 2007, will last up to one year, and is a preparatory period whereby the necessary infrastructure will be installed. Two training centers (Ethiopia and Israel) have been identified; a training program will be developed for MODS at the Hadassah University Hospital in Israel and at the EHNRI in Ethiopia. Trainers will be trained at Central MODS training lab at Israel and regional lab technicians will be trained locally at EHNRI to run MODS. In the 2nd and third phases, the test will be implemented in few regions of the country and its feasibility evaluated.

Anti-Tuberculosis Drug resistance Surveillance in HIV-confected population

TB is a leading cause of morbidity and mortality in Ethiopia. One of the objectives of ensuring effective management of TB is to minimize the development of drug resistance. Surveillance of anti-TB drug resistance is therefore an essential tool for monitoring the effectiveness of TB control programs and improving national TB control efforts.

Reports from the 16th International AIDS Conference in Toronto showed emergence of multiple resistant strains of TB CDC also reported on extensively drug-resistant TB strains resistant to isoniazid, rifampin, and at least three of the six main classes of second-line drugs.

Reliable data about the prevalence of drug resistant tuberculosis at a national scale is not available in Ethiopia. However studies carried out in different areas of the country at different times do report primary resistance to one or more anti-tuberculosis drugs. The Tuberculosis and Leprosy Control Team (TLCT) of the MOH and the National Mycobacterium Reference Laboratory of the EHNRI conducted the first national prevalence of drug susceptibility survey in a representative samples of newly diagnosed smear-positive cases by a population proportionate cluster sampling technique recommended by WHO.. About 40-60% of new TB cases in Ethiopia are HIV co-infected, but there have been few reports about MDR TB in this population. A survey of MDR TB will thus be very helpful for effective treatment of HIV co-infected population. The anti-TB drug susceptibility survey among HIV co-infected patients will be conducted in 2007. The overall goal of the study is to improve the efficiency of TB control and better management of HIV Patients. In the survey, the total number of samples will to be about 1,000. All samples will be collected only from HIV co-infected population. This is based on the following parameters, (an estimated 1% prevalence of resistance to Rifampicin in Ethiopia, a 95 % confidence limit around the prevalence, a precision of 1 percentage point and an additional 13% to account for loses). A minimum of thirty diagnostic centers will be randomly selected from a list of all diagnostic centers in the country using cluster-sampling methods. Before sample collection, a training workshop will be provided for thirty health facilities staff that will be involved in the survey. Positive sputum specimens will be collected from eligible patients at the health facilities and transported within five days to the National Reference laboratory for culture and susceptibility testing. Culture will be done using Petroff's methods. Identification of Mycobacterium tuberculosis complex (MTC) will be according to the standard methods followed at EHNRI. The drug susceptibility test will be performed according to a simplified variant proportion method of Canetti et al. The susceptibility of the strain for Isoniazid, Rifampicin, streptomycin and Ethambutol will be tested since these drugs are used in the TLCT. Drug concentrations used in LJ medium will be according to the manufacturer guidance.

Quality control will be organized to detect system errors and to improve compliance with the survey procedures. It will be applied to all essential elements of the survey including, the sampling, the clinical information, previously treated patients, and the laboratory techniques used at the peripheral level and at the National TB Reference laboratory at EHNRI. To ensure that results of susceptibility testing were reliable and comparable, internal and external; quality control of susceptibility testing was carried out during the survey. For international comparison and acceptance the quality control of the drug susceptibility testing will be linked to a WHO Supranational Laboratory RIVM Bilthoven, the Netherlands. The survey will be conducted by EHNRI and TLCT /MOH with the support of PEPFAR Ethiopia.

Overall the survey on the prevalence of anti-TB drug resistance will involve three major operational issues: program management (logistics, training, collection of clinical information, supervision of survey); laboratory techniques (drug susceptibility testing, proficiency testing, quality assurance); and epidemiology (sampling, data entry and analysis).

plus ups: "Important interventions in TB/HIV collaboration are screening HIV+ persons for tuberculosis, DOTS provision for active TB cases and INH prophylaxis for cases where active TB is ruled out. In Ethiopia however, where TB diagnosis relies mainly on sputum smear examination, physicians are hesitant to put HIV+ clients on INH prophylaxis due to fear of inadvertently treating active TB cases suboptimally with a single drug that potentially could lead to development of resistance to one of the most powerful anti-TB drugs. In resource-poor settings, sputum smear microscopy has been shown to be a simple cost-effective means of diagnosing pulmonary tuberculosis (PTB). Unfortunately, with emergence of HIV, sputum smear microscopy has become less reliable and may be negative in up to 40% of PLWHA with PTB . Despite efforts to improve the yield of smear microscopy through concentration, sputum induction, or quality control measures, it remains a sub-optimal test for HIV-infected individuals.

" "In Ethiopia, more than 50% of reported TB cases are smear negative and extra pulmonary. There are no culture or biopsy services and chest x-ray facilities are inadequate. A PEPFAR supported targeted evaluation of 2005/6 revealed the importance of strengthening TB diagnostic capacities, especially the need to make available sputum culture and chest x-ray facilities in areas serving large number of HIV+ patients. It is also important to assess the feasibility of introducing histology services for extra pulmonary TB diagnosis. " "Description of Activities:1) Only one center in the country currently does mycobacterium culture, the Ethiopian Health and Nutrition Research Institute (EHNRI); therefore, strengthening the current EHNRI TB culture facility and expanding the service to other five regions will be undertaken. Regional TB laboratories will be renovated and equipped for TB culture; laboratory technicians and microbiologists will be trained on culture diagnosis of M.TB. 2) Quality control for sputum AFB examination will be established and onsite training given to laboratory technicians to improve the AFB examination 3) EHNRI will also work with JHU to assess the feasibility of introducing Fine Needle Aspiration (FNA) and histological examination in selected hospitals in Addis Ababa. A number of health centers where physicians are available will be included in the assessment. Details mechanisms will be developed after discussions with relevant stakeholders, including pathology departments of selected referral hospitals. "

Targets

Target Target Value Not Applicable Number of TB patients who are tested for HIV among the  registered TB patients Number of service outlets providing treatment for tuberculosis (TB)  to HIV-infected individuals (diagnosed or presumed) in a palliative care setting Number of HIV-infected clients given TB preventive therapy  Number of HIV-infected clients attending HIV care/treatment  services that are receiving treatment for TB disease Number of individuals trained to provide treatment for TB to 250
HIV-infected individuals (diagnosed or presumed)

Table 3.3.07:

moved to TB per program review

Laboratory Quality Assurance Program

This is a continuing activity from COP05 and COP06. As of April 2006, EHNRI received 100% of COP06 funds and is on track according to the original work plan. We have increased funding based on the achievements from COP05 and partially COP06.

ENHRI is the technical arm of the MOH responsible for providing guidance to laboratory services; the lead institution in coordinating laboratory programs in Ethiopia with a national plan to support HIV laboratory services.

In COP05 and 06, EHNRI focused on improving laboratory services through support to the National HIV surveillance program, establishing laboratory methods , developing standard training curricula, training laboratory personnel in HIV testing and treatment monitoring conducting related research on TB/STI/HIV drug resistances and evaluation of diagnostic technologies. These activities have been implemented in collaboration with MOH, CDC, APHL, and ASCP. EHNRI played a major role in improving the quality of the 2005 National ANC based HIV and Demographic Health Survey (DHS). Implementation of the quality assurance program for HIV serological screening was a further success. The National HIV drug resistance survey was accomplished in partnership with CDC and the Israeli National HIV Reference Laboratory. Because of this effort, Ethiopia was one of the few African countries to complete national HIV drug resistance threshold survey.

Early infant diagnosis of HIV was established at the National HIV Laboratory, EHNRI. Procedures for HIV pro-viral DNA PCR detection from dried blood spots has been validated and piloted at selected ART sites. Preparations are underway to set up testing at six regional Reference laboratories.

The National HIV Laboratory renovated by PEPFAR Ethiopia is intended to serve as the national model and center of excellence, and has started the preliminary laboratory work. To maximize the support and implement the national quality assurance program, to track HIV drug resistance and TOT based training, EHNRI is working closely with PEPFAR Ethiopia, ASCP, and APHL.

In COP07, EHNRI will provide the national leadership in strategic planning, laboratory policies, guidelines, SOPs, training, integrated services and testing, and ensure implementation of laboratory quality assurance, including the National External Quality Assurance Scheme (NEQAS) program to Specialized Referral Hospital Laboratories (SGRHL), Regional Laboratories (RL) and Regional Hospital Laboratories (RHL). The national HIV laboratory will be internationally accredited and will serve as a national center of excellence and the East African regional reference center in drug resistance monitoring, evaluation of appropriate testing technologies, provision of specialized and advanced diagnostic services. It will also serve for TOT based in-service training of laboratory science professionals.

The COP07 activity plan will also focus on the continuation and expansion of COP06 programmatic areas: quality assurance program; training; diagnosis; establishing tiered laboratory services and referral linkages. Support to the eight regional laboratories; (Addis Ababa, Adama, Nekmet, Bahir-Dar, Dessie, Awassa, Mekele and Harar).will be technical and financial (50% of EHNRI's budget). Funding will be divided between the eight laboratories depending on the the relative volume of work performed ,, especially the number of ART Hospital Laboratories under each Regional laboratory. With EHNRI support the regional labs will also support complex tests that hospital labs cannot perform, and provide suitable referral based on test results.

The Institute will continue supporting the establishment and strengthening of laboratory standards and closely work with the eight regional laboratories Together they will strengthen the tiered laboratory services from health center to district hospitals, from district to zonal/ regional hospitals, to reference laboratories. Regional laboratories will support routine the quality assurance and control plan for VCT, diagnosis of OI and laboratory monitoring of ART at hospital and health centers. The regional laboratories will report on all hospital and health centers every three months. Quality control materials (proficiency panel) will be distributed to sites twice yearly. External quality assessment including site visit reports and proficiency panel test results will be regularly communicated

to sites. Regional laboratories will also report their activities to EHNRI quarterly.

EHNRI will continue its training activities, with more emphasis on TOT of laboratory personnel on rapid HIV diagnosis, monitoring of ARV therapy, laboratory equipment maintenance and laboratory quality management system, TB and OI diagnosis and HIV surveillance. EHNRI will ensure that standardized training modules are used for regional and site-level training programs supported by Regional Reference laboratories and US universities.

The National HIV Laboratory will provide referral diagnostic services for HIV/TB/STI drug resistance and EQA including HIV DNA PCR for infant diagnosis, CD4, hematology, and chemistry tests. The regional laboratories will also provide referral-testing services (CD4, Viral load and DNA PCR) and EQA services to hospital and health center laboratories including EQA for HIV rapid testing.

EHNRI will support National ANC based HIV surveillances in 2007. The HIV drug resistance survey conducted in Addis Ababa in 2005 will also be expanded to include other major regional cities in the country. Following on to earlier achievements in the drug resistance threshold survey, EHNRI will formalize its relationship with the Israeli National HIV Reference Laboratory for establishing HIV drug resistance and genotyping technologies, technology transfer and optimum local capacity development. EHNRI will also collaborate with Hadassah University in Jerusalem and the University of California, San Diego (USCD) for sentinel viral load and HIV drug resistance monitoring associated with treatment failures in hospitals and health centers.

PLUS UP FUNDING: The Ethiopian Health and Nutrition Research Institute (EHNRI) supports laboratory trainings, external quality assessment (EQA) in HIV diagnosis and monitoring of treatment at hospitals and health centers which provide HIV care and treatment services. WHO/CDC developed HIV rapid testing training modules to be implemented at all levels through regional and decentralized training schemes. A total of 800 counselors/lab technicians and other mid-level health professionals involved in HIV testing and counseling will be trained. To ensure HIV testing quality, EQA schemes will be implemented at all HIV testing sites. Support will include the following major activities: (1) roll out of regional and facility level trainings in partnership with regional laboratories, covering all primary health care centers providing HIV testing; (2) on-site follow up and supervision of 800 testing sites; (3) preparation and distribution of proficiency testing panels by EHNRI and regional laboratories to all testing sites; (4) retesting selected specimen samples in a reference laboratory to assess testing quality; and (5) evaluating and monitoring the training effectiveness and EQA program in improving the HIV testing. EHNRI will implement the scale up of training and EQA program at all levels in partnership with eight regional laboratories (Addis Ababa, Adama, Awassa, Harar, Bahir-Dar, Dessie, Nekemt, and Mekele). Overall, the national roll out training on HIV rapid testing and EQA program will significantly contribute to scaling up the HIV/AIDS care and treatment program, to achieving national targets , and to improving the quality of HIV testing services at all levels.

Equipment Maintenance and Technical support for Laboratory services

In COP05 and COP06, technical assistance including maintenance of equipments, inventory laboratory management and on-site practical trainings, follow up and supportive supervision were provided to all ART hospital laboratories. In COP07, the technical support will continue and also expanded to new sites which initiated ART services including all health center laboratories.

Maintenance services will be provided for major laboratory equipments at all 131 hospitals, 240 health centers and eight regional laboratories. Preventive maintenance and calibration of major equipments including centrifuges, FACScounts, hematology and chemistry analyzers will be provided quarterly at all sites where the equipments are installed. Broken machines will be repaired and support parts changed to prevent/ minimize service interruption. Technical assistance will include maintenance and troubleshooting of laboratory equipments (fridges, freezers, microscopes, incubators, autoclaves, chemistry analyzers, hematology analyzers and FACScount machines) at all ART hospitals and health centers.

Technical support includes inventory and laboratory management for maintenance of clinical laboratory services and ensures laboratory standards are implemented at all ART hospital, health center and VCT laboratories.

Technical assistance will be provided in the reviewing existing laboratory operating procedures, recording and reporting at facility levels. Assistance will also include production of laboratory stock management tools, disseminated for use at selected sites; implement a system of scheduled requisitioning of laboratory reagents and test kits and other supplies and institute a quarterly reporting system for laboratory commodities consumption and stock status.

Technical assistance will also include on job training in test procedures, preventive maintenance, and functional and structural organization of laboratory, specimen management, data recording, and reporting.

Specimen Management and Transport Services

During COP05 and 06, CDC Ethiopia supported the laboratory monitoring of ART. Laboratory supplies, including diagnosis and monitoring reagents, were purchased and distributed to laboratories for the diagnostic and monitoring tests. Referral testing services were provided for hospital labs which did not have the required machines. Similar referral testing services where specimens were transported to the next level or central facilities had been effectively used in the initial implementation of ART. Since most health centers lack most basic equipment, referral-testing services are extremely important. In COP06, specimen transport services had been provided to peripheral hospitals; in COP06, health centers will be similarly assisted. In order to offer laboratory-monitoring services (CD4, and other basic tests) for PLWHA, including children and pregnant women a sample referral system will be established. Clinical samples will be transported from the outlying clinics to referral laboratories where equipments and human resources are available.

In COP07, the service will expand and include all peripheral hospitals and health centers that initiated ART. To improve the quality of care and treatment, standardized laboratory services must be provided to all patients at hospitals and health centers. The quality of laboratory services rendered should also be maintained. To enroll patients and monitor the efficacy of ART, basic and advanced laboratory tests are required: CD4, biochemical and hematology profiles at specified period of time as per the Guidelines for ARV use in Ethiopia.

To achieve the targets, scale up of ART services at health centers is planned. However, most of the health centers and some peripheral hospitals involved in ART implementation do not have a capacity to support laboratory services. Flow Cytometry for example, to measure CD4 cell counts, is beyond the scope of what can be implemented at health centers. In addition, some clinics do not have the basic equipment for routine CBC, differential, clinical chemistry tests.

The logistic support for referral testing services will be a major undertaking. The specimen transport and transfer system will be supported through courier system. This activity will be contracted to a non-governmental agency that has a track record in the management and transport of clinical sample from primary health centers to regional hospital or referral laboratories. This includes transport of specimens and results to and from health centers to the next level hospital or regional laboratories and/or to National HIV Reference laboratory The lab tests done at the hospital laboratories are returned within two days. It is anticipated that the turn-around time from health center to test site back to the clinic will be 2-3 days. Special transport services will be organized in transporting samples. The agent and the clinic personnel will fill patient tracking numbers and tracking sheets during receipt and delivery.

Samples will be collected in test-specific containers that already contain any necessary preparation reagent. After the samples are collected, the laboratory request form is included in the collection container, placed in a cooler, and transported. All the regular samples are transported in cool gel packs in a temperature controlled thermacol box. To maintain integrity of samples the local samples are delivered within a day using cargo or courier mode. The responsible contractor or agent will receive the samples from the peripheral clinics and transport to the nearby hospital and/or regional laboratories following guidelines and regulation on specimen management and transport. The samples for testing CD4, chemistry and hematology tests will be coordinated and transported at a regular time interval. Clinical samples will be transported from 240 health centers and 25 peripheral hospitals once or twice a week and transported to the testing site (zonal/woreda or regional hospital laboratories). The results will be returned within two day of the tests done. Tunaround time will be 2-3 days

Sample transport from the clinics to hospitals will be coordinated with health center clinics and laboratories on specific days in order to make the most efficient use of limited laboratory equipment and staff. Specific training on specimen management, transport, and storage, recording and reporting will be provided. The specimen management,

transport, and referral system will be followed using the strict guidelines and Standard Operating Procedures (SOPs) developed by the National Reference laboratory, EHNRI and PEPFAR Ethiopia.

In COP07, this activity will expand the laboratory information system to 26 sites to support operations and quality assurance activities in EHNRI, regional laboratories and PEPFAR supported ART hospital laboratories. It will enable sites to have efficient data and report exchanges. To achive this, the following expansion will occur: 1) procurement of 78 additional LIMS software licenses for 26 sites, 2) procurement of 26 barcode printers, 78 barcode readers and 52 barcode lables, 3) training of 52 laboratory technicians and 26 receptionists in LISM, 4) procurement and provision of 78 computers and accessories, 5) design and implementation of a peer-to-peer network for selected regional and hospital laboratories, 6) installation and configuration of LIMS in selected regional and hospital laboratories, linking the hospital laboratories via dial-up with their respective regional laboratories, and regional laboratories with EHNRI reference laboratory, 7) installation of telephone lines into regional and hospital laboratories for LIMS successful implementation, 8) continue technical support to COP06 funded LIMS sites, 9) local travel for technical support and international travel for experience sharing with APHL facilities on LIMS.

None provided.

Table 3.3.13: