Cricetomys sp.

Sokoine University of Agriculture

The potential for reducing tuberculosis burden in

Sub-Saharan Africa using trained African giant pouched rats (Cricetomys sp.) Georgies Mgode (PhD) Sokoine University; APOPO-TB Detection Centre, Morogoro, Tanzania

Introduction



Diagnosis of TB in Sub-Saharan Africa by smear microscopy is a major challenge Trained rats can detect TB rapidly and accurately Highly developed sense of smell than all mammals Detects 28 % of smear negative, culture positive TB 44% increase in TB case detection when rats are used

Training of the rats to detect TB in sputum Newborns In nests with mother Opening of eyes after 4 weeks Habituation/socialization 6 weeks-old rats

Habituation and click training 8 weeks-old rats

11 weeks-old rats 14 weeks-old rats

1 hole sniffing and teaching indication 3 hole sniffing and teaching discrimination 10 holes with multiple samples

26 weeks-old rats

Life-span in captivity: 6-8 yrs

Objectives: the reliability of rats to detect TB  Ability of rats to discriminate clinical sputa with different microbes (Mtb, NTM and non-mycobacterial species).  Ability to discriminate cultures of Mtb from other mycobacteria and respiratory microorganisms.  To identify the Mtb-specific volatiles detected by trained rats for TB diagnosis. • Presence of Mtb-specific volatiles in other mycobacteria and other pulmonary pathogens. • Olfactory detection/discrimination of Mtb-specific volatiles from shared volatiles by rats.

1. DISCRIMINATION OF CLINICAL SPUTA WITH 

MTB, NTM

AND OTHER RESP-TRACT MICROBES •Aliquots of clinical sputa cultured and the remaining tested by 10 trained rats.

Mycobacteria-related pathogens (Rhodococcus, Nocardia and Streptomyces) from detected sputa - FURTHER ANALYSIS

2. Detection rates of clinical sputa with Mtb, NTN and other respiratory tract microorganisms …

• > 28% of smear-negative TB is detected by rats • Sputa with Rhodococcus, Nocardia and Streptomyces detected due to dormant Mtb ?

2. Detection of sputa with other microorganisms  associated with Mtb

3. DETECTION OF CULTURES OF MTB, OTHER MYCOBACTERIA AND MICROORGANISMS

•Rats detects Mtb cultures better than NTM (P < 0.001, Fisher’s exact test).

•The detection of Mtb is based on age of cultures. Rats detection curve

Mtb growth curve

4. Mtb volatiles detected by rats • 26 volatile compounds produced by Mtb and other microorganisms in 5 types of media and growth stages. • 13 volatiles (50%) are specific to Mtb, other 13 are found in Mtb and other microorganisms

4. Clustering of Mycobacteria and other respiratory microbes based on their volatiles

Significant clusters consist: •Mtb only •Mtb and NTM •Mtb, NTM and other microorganisms.

5. Olfactory detection of Mtb volatiles by rats 1. Mtb-specific volatiles tested individually by rats

Methyl nicotinate

Methyl para anisate Ortho phenylanisol

Rat-negative

5. Olfactory detection of Mtb volatiles by rats 2. Mtb-specific volatiles tested in combination

3 others Methyl nicotinate

Methyl para anisate Ortho phenylanisol

Rat-positive • Rats detect a combination of 6 Mtb-specific volatiles than individual odour compounds (P = 0.001, Fisher’s exact test).

5. Olfactory detection of Mtb volatiles by rats Rats detect mild concentration of Mtb odour than higher and lower concentrations (P = 0.001, Fisher’s exact test).

Un-specific volatiles found in Mtb, NTM and other microorganisms are not detected by rats (in over 96 presentations).

6. SENSITIVITY, SPECIFICITY AND ACCURACY OF RATS TO DETECT TB IN CONTROL CLINICAL SAMPLES

Rats detected 118 of the 119 TB positive control sputum samples

•Rats detected all 7 TB-positive sputa (100%) in 16 out of 17 presentations, 6/7 TBpositive sputa (85.7%) once. •Rats detected 61 out of 785 negative control sputum samples (false positive) in 17 presentations (days). Average false positive rate = 7.8%. •The sensitivity of detecting typical TB-positive sputa = 99.2%.

•Specificity = 92.2%. •Accuracy = 93.1%. • Rapid detection of TB cases (70 samples /32 min) vs microscopy (20 sputa/ 8hrs).

CONCLUSIONS •Trained rats can reliably and accurately diagnose TB (target a blend of Mtb-specific odour) • > 28% of smear-negative, culture positive TB detected by rats. • Smear-negative sputa detected by rats likely contain low concentration of Mtb not detected by microscopy and or dormant Mtb not culturable in conventional medium. • Rats increase case detection by 43-44% (second line screening after microscopy). •High adaptation of Cricetomys rats to diverse climatic conditions and the longer life-span (8 years) makes rats suitable for control TB in most Sub-Saharan Africa. •Efforts needed to expand the use of this affordable technology in other African countries with a high burden of TB. Tanzania and Mozambiques findings very promising. • Mass screening could be achieved by using rats to reduce work load in TB clinics, reduce misdiagnosis and delayed diagnosis.

Acknowledgments •MPIIB, Berlin

Doris Lazar January Weiner

Stefan H.E. Kaufmann •Institut für Organische Chemie, TU-Braunschweig

Thorben Nawrath Stefan Schulz UBS-OPTIMUS FOUNDATION

• SUA-APOPO Bart Weetjens Christophe Cox Maureen Jubitana Negussie B Mahoney A; Christiaan M Rat-trainers & Technicians •National Inst. for Medical Research (NIMRMuhimbili, Tanzania) •National TB & Leprosy program, Tanzania •DOTS Centres – Dar es Salaam, Tanzania