+ Quality Assessment of Malaria Diagnosis on the Thai-Burma Border Jaime Moo-Young Noon Conference, Harborview Medical Center June 1, 2010

Background: Malaria Situation in Thailand Overall incidence decreasing for past 20 years Thai-Burma border still area of highest transmission Tak Province has highest incidence rate in country (over 35x national average) Tak Province has highest prevalence of drug-resistant Plasmodium falciparum in the world. Source: Zhou et al, 2005(159)

+ Tak Province and Mae Sot District: A Closer Look Mae Sot: Crossing point between Northern Thailand and Burma Large numbers of refugees and migrant workers crossing border

+ Burmese Migration and Malaria Ruling military junta for over 50 years Ethnic resistance fighting  civil war  human rights abuses Over 2 million refugees fleeing to Thailand within past 10 years Labor and forced evacuation into endemic forest areas Inadequate health care services in remote areas Courtesy of Back Pack Health Worker Team

Mae Tao Clinic Established in 1989 in Mae Sot, Tak Province Founder: Karen doctor and refugee Cynthia Maung Clientele IDPs from Burma who cross border for care Migrant workers and refugees living in Mae Sot Over 100,000 patients seen and treated each year

+ Malaria Diagnosis: How to Meet Best Standards in Remote Field Settings? Microscopy facilities should undergo regular quality control (QC) from a qualified external agency Goal: Sensitivity ≥ 95%  do not miss a positive case, esp. Pf! Specificity ≥ 95%  avoid unnecessary treatment or treatment with the wrong drug, which contributes to drug resistance

Modalities for Diagnosing Malaria ModalityPros Cons Most Commonly used in... Microscopy Inexpensive Can distinguish between malaria species Can diagnose other blood abnormalities Accuracy depends on skill level of microscopists Requires well-trained, long-term staff Requires regular equipment maintenance and quality assurance Any field setting that can maintain adequate microscope facilities and staff * Gold standard for field* Rapid Tests (Paracheck ® ) Very fast Accuracy does not depend on skill of tester Portable, disposable Costly Many false positives Only detects Pf Any remote field setting where good microscopy facilities are not available PCR (polymerase chain reaction) Most sensitive test available Most accurate for species diagnosis and mixed infections Costly and time- consuming Requires sophisticated equipment, trained staff. Too sensitive? Research (genotyping, resistance studies, epidemiological studies)

+ Question: How good is the quality of laboratory malaria diagnosis done by staff at the Mae Tao Clinic, a field setting along the Thai-Burma Border?

+ Study Objectives 1. Determine the sensitivity and specificity of malaria microscopy at the Mae Tao Clinic, using PCR as the gold standard 2. Compare the sensitivity and specificity of malaria microscopy when performed by Mae Tao Clinic staff with that performed by experts at the Thai Ministry of Public Health (MoPH), Bangkok 3. Identify suggestions for improvement of current microscopy practices in the field

+ Methods: Participants Study period: March 5-31, 2007 (4 weeks) Inclusion Criteria: ≥ 5 years old Referred from Adult OPD, Child OPD, or RH OPD History of fevers Exclusion Criteria:  5 years old Follow-up patient Patients unable to give informed consent

Methods: Sample Collection 1. Mae Tao Clinic staff collected PCR sample

Sample Collection (cont.) 2. Mae Tao Clinic staff collected blood smear.

Methods (cont.) 4. Blood smears read and diagnosed by Mae Tao Clinic Lab staff. 5. Patient treated accordingly.

2887 total blood smears taken over sample collection period Total = 339 Selected for QC 423 Smears not selected for QC 203 Smears Selected for QC (32.4%)136 Smears selected for QC (100%) 626 Negative Smears136 Positive Smears 24 slides/PCR sample lost or mislabeled 762 had blood smears and PCR samples successfully recovered 59 patients did not consent 786 patients consented to participate in QC project 845 met inclusion/exclusion criteria1383 did not meet inclusion/exclusion criteria

Sample Analysis back in Bangkok 6. Slides read by Thai Ministry of Public Health laboratory staff, blinded to Mae Tao Clinic results * Results discordant with MTC results were repeated a second time. The second reading was taken as the final MoPH result.

+ PCR Back in Bangkok First Amplification Reaction Product = Plasmodium gene common to all Plasmodium species (1200 bp) Second Amplification Reaction Product: Species-specific gene unique to each different malaria species Pf gene: 205 bp Pv gene: 120 bp Pm gene: 144 bp Po gene: 800 bp Courtesy of Snounou et al, 1993

+ Visualizing PCR Products Po infectionPf, Pv, and Pf + Pv infections Po infection Pm pos. control + Po pos control Pf Pv Pf Pv Pv Pos.control Pv Pf Pv Pf + Pv mixed Pv pos. cont Pf pos. cont Pf pos. control * Results discordant with slide readings were repeated 2-3 times

Results: Frequencies (t = 339) Dx Method NegPfPv Pf + Pv Pf + Pv + Pm Pf + Pv + Po PmPo MTC Microscopy % % % 9 2.7% % 0 MoPH Microscopy % % % 3 0.9% 0000 PCR % % % 9 2.7% 1 0.3% 1 0.3% % Pf = Plasmodium falciparum; Pv = Plasmodium vivax; Pm = Plasmodium malariae; Po = Plasmodium ovale

Errors (out of 339 Slides) Diagnostic Method Total # Neg/Positive Errors Total # Species- Specific Errors Total # of missed mixed infections MTC Microscopy MoPH Microscopy * Using PCR as Gold Standard

Clinical Consequences of Errors Diagnostic Method Untreated Pf Cases Unnecessary treatment of Pf Untreated Pv Cases Unnecessary treatment of Pv MTC Microscopy MoPH Microscopy 10152

Main Quality Indicators (Using PCR as Gold Standard) SensitivitySpecificity Positive Predictive Value Negative Predictive value Mae Tao Clinic Micrsoscopy 90.5%98.9%98.5%93.1% MoPH Microscopy 93.2%98.9%98.6%95.0%

Slide Quality (n = 339) Thick Smear Quality Thick Smear Color Thin Smear Quality Good99 (29.3%)78 (23.0%)110 (32.4%) Average34 (10.0%)147 (43.4%)88 (26.0%) Needs Improvement 206 (60.8%)83 (24.2%)139 (41.0%) Other032 (9.4%)2 (0.6%)

Sources of Error MTC MicroscopyMoPH MicroscopyPCR Staff skill level Slide Quality Recording error No opportunity for “second read” Staff skill level Slide Quality Stain deterioration over time Sample contamination Subpatent parasitemia Misreading of gel

+ Summary of Findings MTC Specificity and Positive Predictive Value are excellent. MTC Sensitivity and Negative predictive value are satisfactory but could be improved (too many false negatives). MTC Skills are very comparable to the MoPH in terms of positive and negative readings, but species diagnosis could be improved. Smear and stain quality appear to be a significant problem.

+ Mae Tao Clinic: Suggestions for Improvement 1. Better slide preparation techniques 2. Both internal and external Quality Control (QC) should continue regularly. 3. More inexperienced staff members should have closer supervision. 4. Efforts should be made to liaise with other local NGOs and governmental organizations in order to streamline standards and make each border lab facility part of a network.

+ Conclusions Despite some shortcomings, the Mae Tao Clinic has been able to implement an adequate malaria microscopy facility in a resource-limited field setting. Improvements in Giemsa stain quality, smearing techniques, and washing slides would improve baseline accuracy in reading blood smears. The Mae Tao Clinic should be able to serve as a training hub for clinics across the border, provided they continue to monitor their services via regular QC and make the suggested improvements within their means. *A standardized, reliable network of monitoring, evaluation, and quality control strategies should be implemented among ALL health service providers in this highly endemic region