DIRECT MICROSCOPY Summary case for inclusion as Essential Diagnostics Good afternoon all. Today I am going to talk to the case for inclusion of direct microscopy on the EDL Direct microscopy has broad applicability, I will focusing on the 4 serious fungal opportunistic infections that are estimated to cause the highest burden of disease in advanced HIV – CM,Pneumocystis pneumonia, Disseminated Histolasmosis and Chronic pulm aspergillosis post TB Essential Diagnostics for Advanced HIV Disease 11 April 2018 Kampala Uganda Charlotte Sriruttan National Institute for Communicable Diseases (NICD), South Africa

DEFINITION ADVANTAGES DISADVANTAGES Direct microscopic examination of clinical specimens Traditional, phenotypic method – detection of organisms based on morphology Technicians/technologists Rapid availability of results (2-4 hours). Faster than culture if positive Detection of fungi directly in clinical specimens – some morphology highly characteristic of certain infections Visualisation helps confirm that organism is in the sample rather than contamination Additional infections can be identified Accessibility - simple equipment needed (light microscopes, slides, basic stains available in almost all health labs) Cost Not definitive diagnosis Dependant on personnel with specific mycology skills Sensitivity varies with agent, source and quality of specimen and observer experience Viability Difficult to quantify and replicate Electricity supply for microscopes Not an option where no lab facility DEFINITION ADVANTAGES Definition : microscopic examination directly on clinical specimens from usually sterile sites such as CSF, aspirate fluid, bronchoalveolar lavage, touch preparations, tissue sections Traditional or classic method for detection of organisms based on morphology Most often performed by technologists/technicians across different levels of health labs – esp in LMIC and at level 1 labs (district/state/provincial labs) where pathologists often not a resource Rapid TAT: Direct microscopy result often influences treatment with potential to improve outcome Morphology of fungi that may guide rx decisons– yeast or yeast like org or hyphae (septate/aseptate) Guatemala project data presented yesterday showed 10% of patients with advanced disease had coinfections (TB plus fungal or dual fungal OI) with associated high mortality Solar charged batteries for microscopes may be an option DISADVANTAGES

1. Cryptococcal meningitis SPECIMEN TYPE DIAGNOSTIC TEST PERFORMANCE CHARACTERISTICS CSF CrAg LFA India ink preparation 100% sensitivity, >99% specificity Up to 80% sensitivity The case for optimal dx for CM i in WHO 2018 cryptococcal disease guidelines – CrAg LFA is preferred dx test, however where not available India ink is recommended This paper LID 2017 by colleagues here – “ improvement of fungal dise identification and mx” highlights india ink should be the alternative in low income countries at level 1 labs

2. Pneumocystis pneumonia SPECIMEN TYPE DIAGNOSTIC TEST PERFORMANCE CHARACTERISTICS Respiratory fluids PCR Microscopy - Immunoflourescence (IF) - Calcoflour white Diff-Quik GMS is more sensitive than immunofluorescence with sensitivity ranging from 90-100% Direct IF sensitivity of 86-97% Until PCR is available on a simpler cheaper platform, microscopy is the only other dx assay that is feasible in resource-limited settings In this study pubihed in 2004, looking at detection of p.jirovecii by 4 staining methods , the Calcofluor white and GMS stains had the best parameters for routine use in a clinical laboratory. Although these assays were less sensitive than the immunofluorescent assay, they were highly specific and had more than acceptable positive and negative predictive value Good correlation with diff-quik and IF as discussed by our colleague in the AIDS and respiratory case scenario yesterday

3. Disseminated histoplasmosis SPECIMEN TYPE DIAGNOSTIC TEST PERFORMANCE CHARACTERISTICS Peripheral blood smear Giemsa or Wright’s stain : small intracellular yeasts ? but higher rates of positive cultures 70% Histo ag detection – ELISA test has good performance but not currently readily available Histo ag LFA is needed Culture – long, lab safety issues – not EDL Stains poorly with Gram stain DDX : Emergomyces/ T.marneffi important OI in AIDS pts in certain geographic areas - but may seen fission wall (belt)/ other small Candida/ Difficulty in dx in the African setting is highlighted in this paper written by colleagues in the audience and demonstrated by the last clinical case yesterday – Histoplasma detected on peripheral blood smear, then BM and culture result

4. Chronic pulmonary aspergillosis (CPA) SPECIMEN TYPE DIAGNOSTIC TEST PERFORMANCE CHARACTERISTICS Sputum Microscopy Microscopy of sputum may show hyphae morphologically consistent with Aspergillus spp. If present, this finding is most consistent with CPA or Aspergillus tracheobronchitis As per the EID paper in progress : Case definition for CPA in resource limited settings generated from last GAFFi workshop In the correct clinical and radiological context, direct microscopy has a role if aspergillus ab tests are negative 63.     Langridge PJ, Sheehan RL, Denning DW. Microbial yield from physiotherapy assisted sputum production in respiratory outpatients. BMC Pulm Med 2016; 16: 23,016-0188-2. Proposed algorithm If the Aspergillus IgG antibody test is negative, then sputum microscopy for hyphae or fungal culture should be performed. If the sputum microscopy for hyphae or fungal culture is negative, then other diagnoses should be considered, such as atypical mycobacterial infection or endemic fungal infection. If the sputum microscopy for hyphae or culture is positive, or if serum Aspergillus antibody test is positive, then CPA is confirmed and treatment with itraconazole or voriconazole is advised. Case definition for CPA in resource limited settings - EID in progress Proposed algorithm If the Aspergillus IgG antibody test is negative, then sputum microscopy for hyphae or fungal culture should be performed. If the sputum microscopy for hyphae or fungal culture is negative, then other diagnoses should be considered, such as atypical mycobacterial infection or endemic fungal infection. If the sputum microscopy for hyphae or culture is positive, or if serum Aspergillus antibody test is positive, then CPA is confirmed and treatment with itraconazole or voriconazole is advised.

SUMMARY Cryptococcal meningitis CSF : India ink if no CrAg LFA DISEASE DIRECT MICROSCOPY Cryptococcal meningitis CSF : India ink if no CrAg LFA Pneumocystis pneumonia Respiratory fluids : Microscopy if no PCR Histoplasmosis Peripheral blood smear : Giemsa/Wright stain may give rapid presumptive dx if no antigen test available Chronic pulmonary aspergillosis Proposed algorithm : sputum microscopy has a role if Aspergillus IgG antibody test is negative

To further make the case , this NEJM EDITORIAL JUNE 2016 –perspective piece includes microscopy in list of lab tests that are required for use of anti- infectives on WHO EML

CONCLUSION “The 4th 90 %” with focus on reducing deaths today direct microscopy has a key role in rapid diagnosis currently low hanging fruit if optimise access to stains, training, QA, harnessing technologies where ELISA/PCR unavailable and LFD technologies being developed/data Direct microscopy is essential, but can it be considered for EDL? Optical brighteners -test can be rapidly performed and read, which enhance detection of fungal structures in respiratory and other specimens increasing sensitivity– easier detection/enhance structures eg easier to see if hyphae are septate Elearning modules eg microfungi.net QC/QA and calibration and method validation as we heard this morning Sending samples for quality assessment to test all processes – challenging but as mentoned by Dr Arunaloke work is is progress (ISHAM) Essential but as already available/not IVD how can it be considered for EDL