Minutes of 17 th RNTCP National Laboratory Committee held ... · (2) Update on IRL strengthening by...

Minutes of 17th RNTCP lab committee

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Minutes of 17th RNTCP National Laboratory Committee held on 7th July 2009 at New Delhi The 17th RNTCP National Laboratory committee was held at conference hall of New Delhi TB centre on 7th July 2009. Agenda is annexed at annexure-I. List of Participants is annexed at annexure-II. The meeting was chaired by Dr. V M Katoch, Secretary, Dept of Health Research, Govt. of India and DG, ICMR. DDG (TB) welcomed the participants and presented the implementation status of the recommendations made by the previous lab committee meeting held on 7th March 09. The support to the programme from all National Reference Labs was appreciated. The National Reference Laboratories were urged to be proactive in implementing the decisions of the laboratory committee, especially to ensure the uniform standards and methodology in Culture and DST. The following agenda items were discussed.

(1) The results of validation phase of RNTCP and FIND collaborative project on Line Probe Assay (Genotype MTBDR plus) for diagnosis of Multi-drug resistant TB [MDR-TB]). Standard operating procedures for the demonstration phase of the study.

(2) Update on IRL strengthening by PATH (3) Global Laboratory Initiative/Green Light Committee (GLI/GLC) mission to Andhra

Pradesh, Haryana, Orissa and Uttarakhand for assessment of MDR-TB diagnosis and treatment aspects, including the laboratory preparedness for uptake of newer technologies for MDR-TB, specifically under UNITAID support.

(4) The minimum requirements for negative air pressure rooms; and requirements for the molecular diagnostic facilities at Culture and DST facilities.

(5) Reorganizing the National Reference Laboratory (NRL) activities under the RNTCP national laboratory network.

(6) The action-plans for the recommendations of World Bank short-term consultant who visited IRLs in Jan – March 09.

(7) Update on C & DST laboratory accreditation status of IRLs, Private sector and Medical college laboratories, and update on the smear microscopy EQA activities- OSE visits of NRLs.

Committee Recommendations All NRLs would make weekly telephonic calls with the (a) director and (b) microbiologist of the Intermediate Reference Laboratories, regarding the status of lab and actions required for corrective measures. The laboratories where the accreditation process has been delayed assume the first priority.

⎯ A brief of telephonic discussion be prepared and sent to the concerned IRL with copy to STO and CTD.

⎯ Based on the information from NRL, CTD would take up the issues with IRL and STO.


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► FIND & RNTCP collaborative projects FIND made presentations on status of RNTCP-FIND collaborative projects in India. The committee made following recommendations/observations:

Molecular Line Probe Assay (LPA) 1. The results of Line Probe Assay evaluation phase (annexure III) were

discussed. Sensitivity and Specificity for Rifampicin detection, after reconciling the results with DNA sequencing of rpoB locus, was 96% and 99%, respectively. Sensitivity and specificity for detection of isoniazid, after reconciling results to KatG and InhA loci was 72% and 98%, respectively. The committee agreed that the results of evaluation phase were as per the previous scientific reports from India and elsewhere. It is recommended that the study may proceed further with the demonstration phase to answer the challenges of using the LPA for the patient management under the programmatic conditions.

2. Secretary of Health Research and DG, ICMR encouraged the FIND team to, optionally, validate the LPA test with sputum specimens transported using Cetyl Pyridinium Chloride. This would obviate the need for the cold chain for sputum transport.

3. The SOPs for the demonstration phase were briefly discussed and approved. It was recommended that to resolve any the LPA-LJ media discordance in rifampicin susceptibility by testing at JALMA (sequencing) and LRS (repeat DST on a different culture system).

4. It is to be ensured that adequate trainings of staff and sufficiency of Cat IV drug supply is available before starting the demonstration phase.

Liquid culture systems 1. FIND presented limited evaluation data on Bactec-MGIT 960 liquid

culture system. 2. Demonstration studies for MGIT 960 would be conducted at two sites-

STDC, Ahmedabad and LRS Institute, Delhi. The objective of the demonstration study is: to assess the operational feasibility of use of automated liquid culture and DST system in RNTCP culture and DST laboratories. Specific outcomes will be specified in the demonstration study protocol.

3. Up gradation of IRL at Hyderabad, undertaken by PATH, is expected to be completed in 3-4 months. The option of shifting this demonstration site to any NRL, where negative air pressure facilities are available, may not be suitable for obtaining data on programmatic evaluation of the MGIT 960.

4. The requirement for appropriate bio-safety level to handle the infectious aerosols of Mycobacterium tuberculosis was discussed. In addition to WHO BSL-2 requirements, negative air pressure rooms, at the minimum, are required for manipulating of liquid cultures.

LED-Fluorescence Microscopy: 1. The microscopes evaluated under this study (Primostar iLED) are dual

function microscopes- both for Fluorescence Microscopy and bright field light microscopy.

2. It was again reiterated that once the present study is completed and results evaluated favourably, the replacement of the non-functional/condemned RNTCP microscopes should be done with LED-based illumination light microscope. Where electricity-supply is particularly erratic, replacement or revision of microscopes with LED illumination (battery-backup enabled) would be pursued. Also, where microscopy loads are high (such as certain


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DTC-DMCs and DMCs under medical colleges) Fluorescence microscopy option would have advantages; and in cases where electricity interruptions in the work are expected (certain peripheral DMCs) the battery operated light microscopy option could be advantageous. The technology would be used accordingly.

► PATH technical assistance to identified IRLs

PATH, with an ASM microbiologist, made an assessment visit to six IRLs- Jharkhand, Karnal, , Lucknow, Orissa, Uttarakhand and West Bengal. Since the labs were not yet accredited by the NRLs, several technical and operational deficiencies were noted during the visit. CTD has communicated these aspects to the respective states for necessary corrections.

1. PATH would coordinate with the NRLs during their next visits, and obtain the necessary support

2. With regard to Karnal laboratory, the following actions were decided:

⎯ PATH will assist the LRS Institute to make a technical brief of present status along with all recommendations of the previous visits as annexes.

⎯ LRS communicate the same to STO and IRL with a copy to CTD.

⎯ Based on the information from NRL, CTD would take up the issues with IRL and STO and Principle secretary of Health.

3. PATH would visit IRL, Karnal and IRL, Uttarakhand along with technical staff

from the respective NRLs of the states, for ensuring the corrective measures.

4. The on-site orientation/ training for the IRLs (Karnal, Ranchi & Dehradun) will be initiated by PATH in coordination with NRLs

► GLI/GLC mission to states of Andhra Pradesh, Haryana, Orissa and Uttarakhand (27th July to 14th Aug, 2009)

The objective of the visit is to assess the ongoing GLC-approved MDR-TB activities and achievements, including laboratory preparedness and adequacy of capacity to implement new MDR-TB diagnosis tools. 1. The minimum requirements (enclosed as annexure IV & V) for negative air pressure

rooms and molecular facilities would be intimated to all states by CTD facilitating the laboratory scale-up plans and uptake of newer diagnostics.

2. The STOs, STDC Directors, and Microbiologists of states which are slated for introduction of newer technologies – LPA and MGIT 960- for the year 2009-10 would be called for a meeting with CTD to facilities action-plan for laboratory up gradation.

3. Funding needs for certain labs without any provision for the negative air pressure rooms would be sought from the World Bank through administrative approval.

►Reorganising the function of NRL activities under the RNTCP laboratory net work

Given the task of scale up of laboratory capacity for Culture and DST, it is felt that respective NRL functions with regard to C&DST responsibilities could be reorganised. The allotment of states to NRLs remains the same and the EQA-OSE and RBRC


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functions remain the same. The following tasks/functions are agreed by the respective NRLs

National Tuberculosis Institute (NTI), Bangalore (1) National level training venue EQA for Smear Microscopy (2) National level Training venue for solid culture and DST. All the three NRL would participate as

faculty during the training. (3) A. Supervise/ oversee up-gradation plans for labs (BSL 3) including technical assistance to all C &

DST labs with support from the national expert group and other NRLs (Subject to strengthening this NRL with provision of national task force with experts). B. Accreditation process -pre-assessment visit, qualification of the lab as per guidelines, pre accreditation visit and accreditation ((Subject to strengthening this NRL with provision of national task force with experts).

(4) Provide support to the programme for second line DST for category IV services

Tuberculosis Research Centre (TRC), Chennai (1) Provision of Technical Assistance to the CTD and all the NRLs (2) Evaluate and perform research related to newer diagnostics (3) Provide EQA for TB Culture DST for the NRLs (4) Provide EQA for public and private labs performing liquid culture based DST (5) Provide support to the programme for second line DST for category IV services

LRS Institute, New Delhi (1) Training for Liquid culture and DST. Faculty from other NRL would participate as facilitators, as

required. (2) Provide services under Plan B whenever required (3) Building up the capacity of LRS for liquid cultures. Provide PT for liquid culture and DST, after

qualifying PT through Antwerp (4) Provide support to the programme for second line DST for category IV services (after building

capacity and undertaking proficiency testing)

JALMA Institute, Agra (1) Provision of training in molecular methods. Faculty from other NRL would participate as

facilitators, as required. (2) Evaluate and perform research related to newer diagnostics (3) EQA for LPA labs (including PT) (4) Provide support to the programme by accreditation of IRLs of states attached, DST support to

these states and for second line DST for category IV services (after building capacity and undertaking proficiency testing)

► Status of accreditation of C&DST labs

NRLs and CTD updated the lab committee with the status of accreditation of C & DST labs and the EQA OSE visits. The following are committee recommendations/observations;

♦ A formal accreditation visit from NRL to IRL West Bengal and IRL Orissa to be carried out by NTI asap.

♦ ICMR has initiated steps to involve its labs at six places for augmenting the programme capacity. These labs are – Bhubaneshwar, Dibrugarh, Jabalpur, Jodhpur, Patna and Port Blair. Training of staff at these places would be conducted at TRC, Chennai. TRC would carry out a visit to ICMR lab at Patna for an assessment.


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♦ ICMR laboratory at Dibrugarh would be visited by PATH along with experts of JALMA and TRC.

♦ Difficulties are perceived in some IRLs with regard to Inspissators. The revised technical specifications for the inspissator (along with suggested suppliers) would be obtained from the JALMA and circulated to other labs.

♦ For DOTS-Plus services, in case Chhattisgarh, Jharkhand, and Uttarakhand IRLs are not accredited in time, the respective NRLs would take up the C & DST for these states, under plan B.

► DRS-activities in Orissa

♦ It is felt that the Orissa STDC laboratory would not be able to undertake responsibilities of DOTS-Plus patients as well as DRS study samples, at the same time. NTI, Bangalore agreed to perform C&DST for DRS Orissa samples at their laboratory. The requisite support for the same would be obtained from the state.

► Number of specimens for culture for diagnosis and follow-up of MDR patients

The committee discussed the number of specimens for the diagnosis and follow-up of MDR TB.

MDR-TB diagnosis (solid media): Two sputum specimens. One of them should be a morning specimen and other is a “spot” specimen under the supervision of a Laboratory staff.

MDR TB patient follow up (solid media): Only one sputum sample (“Spot”) collected under the supervision of a Laboratory staff would be sufficient.

The committee has recommended that brief operational research be conducted to assess the impact of a reduction in the number of follow up sputum specimens from 2 to 1 for MDR-TB patients. This practice was noted to offer many programmatic advantages, and is likely to generate adequate information for monitoring of Cat IV treatment response. All efforts may be made to finalise the data before next lab committee.

► Generic SOPs for C & DST (Solid media):

Generic SOPs for C & DST (Solid media) have been developed by the CTD and are available in the RNTCP website.

• The committee stressed the urgency of implementing the uniform procedures and practises at all IRLs.

• Comments of NRLs and key recommendations of World Bank and PATH assessment teams would be incorporated in SOPs. There is an immediate need to implement the SOPs in all the IRLs, across the country, immediately. If further meetings/workshops are needed, the same would be facilitated at one of the NRLs.

• Accordingly, States would print the laboratory registers for C & DST and SOPs for lab purposes on an uniform pattern to be provided by CTD.

• All 13 labs either accredited/or in the process of accreditation would implement SOPs and report the performance indicators to CTD and NRLs.

• The states and IRLs should ensure that the terms and conditions for the Annual Maintenance Contract ( AMC) of IRL equipments should have all the relevant


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details including the response time required to attend the emergency, provision of a written report by the agencies, penalty for not attending on time etc. NTI is requested to provide a sample format which may be incorporated in the SOPs

► Laboratory assessment report of the World Bank consultant World Bank had tasked a consultant to undertake an assessment of RNTCP IRLs from January to March 09 and made a number of recommendations.

• The recommendations would be circulated to NRLs and respective states.

• NRL would draw actions plans for their respective states in correcting the deficiencies. It would be communicated to CTD before 10th August 2009.

• CTD would ensure the implementation of the action-plan.

***


1

Annexure I

RNTCP17th National Laboratory Committee meeting

07th July 2009 New Delhi TB Centre, New Delhi

Objectives of the meeting • Discussion Results of Molecular Line-Probe Assay (LPA) Evaluation study • Discussion on SOPs for the demonstration study of LPA • Update on the status of accreditation of C&DST labs-implementation of SOPs for

C&DST • Update on EQA for sputum microscopy & DRS activities

AGENDA07th July 2009

09.30 hrs

Agenda items

Introduction DDG(TB)q 1. • LPA based diagnosis of MDR-TB: Results of

validation study • Initiation of LPA demonstration study -SOPs

for demonstration study • Update on FIND projects (Liquid culture, &

LED-FM)

FIND Presentation followed by discussion

2. • Update on PATH Laboratory technical assistance

PATH

3. • GLI/GLC mission to assess laboratory preparedness for MDR-TB treatment services in the states of Uttarakhand, Haryna, Orissa and Andhra Pradesh

• Minimum requirements for Negative air pressure facilities for liquid culture; and requirements for molecular line probe assay

WHO/CTD

4. Discussion: • Restructuring of NRL functions of RNTCP

Laboratory network

CTD & WHO

5. Update on: • Status of accreditation of IRLs, medical college

and other sector labs • Status of DRS surveys –AP,UP, Orissa (inform

earlier to states) • Plan B for states without C&DST facilities:

Uttarakhand, Chattisgarh, Jharkhand

NRLs, WHO, CTD

6. Discussions: • Number of Follow-up specimens for culturing

for MDR-TB patients: One or Two. • Implementation of Generic SOPs for C & DST

(Solid media)- Recording & Reporting • Uniformity in C&DST DST dilutions (Proportion

method-solid media) • Laboratory Assessment reports- Main findings:

World Bank consultant

CTD, NRLs, WHO,

7. • Update on NRL EQA OSE visits (Smear Microscopy) & RBRC activities in the states

NRLs


1

Annexure-II List of Participants

(1) Dr V M Katoch, Secretary, Dept of Health research, Govt. of India, and DG ICMR

(2) Dr L. S. Chauhan, DDG (TB) (3) Dr D. Behera, Director, LRS, New Delhi (4) Dr Prahlad Kumar, Director, NTI, Bangalore (5) Dr. K S Sachdeva, CMO, CTD (6) Dr Vanaja Kumar, TRC Chennai (7) Dr P. Vishalakshi, LRS, New Delhi (8) Dr. R Mandal, LRS, New Delhi (9) Mr Anand, NTI, Bangalore (10) Ms Reena, NTI, Bangalore (11) Dr S. Sahu, NPO (TB), WHO India (12) Dr. Fraser Wares, MO(TB), WHO India (13) Dr Ranjani Ramachandran, WHO-SEARO (14) Dr Puneet Dewan , MO(TB), WHO-SEARO (15) Dr Yamuna, Medical officer, FIND (16) Dr Neeraj Raizada, Associate Medical Officer, FIND (17) Dr Sheena Susan George, Medical Officer, PATH (18) Dr Sarabjit Chadha, WHO-RNTCP Consultant, CTD (19) Dr Ajay Kumar T, WHO-RNTCP Laboratory Consultant, CTD

Annexure III

LPA Validation and Demonstration project

Dr. N. RaizadaFo ndation for Inno ati e Ne Diagnostics Ne DelhiFoundation for Innovative New Diagnostics, New Delhi

1

Parts of the projectParts of the project

1 Initial pilot and proficiency phase1. Initial pilot and proficiency phase2. LPA testing on Direct sputum specimen

1 V lid ti h (Bli d d LPA t ti )1. Validation phase (Blinded LPA testing)2. Demonstration phase (LPA result- Pt

management)management)3. LPA testing on culture isolates with

k DSTknown DST

2

Initial pilot and proficiency testing

3

Initial pilot and proficiency testing (1)Initial pilot and proficiency testing (1)

• After initial LPA training, each site collectedAfter initial LPA training, each site collected sputum specimens from 50 smear + pts for anonymous LPA testing.

• Results were assessed for 1. Negative control validity;2. Successful amplification;3. Internal reproducibility from the same lab; & 4. External reproducibility at more experienced lab

4

Initial pilot and proficiency testing (2)Initial pilot and proficiency testing (2)

• Any site failing proficiency benchmarksAny site failing proficiency benchmarks,a) reviewed practices and b) repeated another proficiency testing roundb) repeated another proficiency testing round c) External experts sent to labs to provide

additional technical support if 2nd failure ofadditional technical support if 2 failure of proficiency

d) Site did not contribute to studies till proficiencyd) Site did not contribute to studies till proficiency testing passed

5

Table: Proficiency results for line probe assay

Proficiency Invalid results Internal External Proficiency testing

round and result

Negative control

Invalid results (number

invalid/total number, %)

concordance (same patient,

same laboratory)*

concordance (same specimen,

different laboratory)*

Benchmark Clean < 10% > 95% > 95%

1stΧ Contam 14/ 48 (29%)

Lab A

1st Χ Contam. 14/ 48 (29%) - -

2nd Χ Clean 15/22 (70%) - -

3rd√ Clean 5/118 (4%) 54/54 (100%) 19/19 (100%)3rd √ Clean 5/118 (4%) 54/54 (100%) 19/19 (100%)

Lab B1st Χ Clean 15/ 100 (15%) 36/37 (97%) 17/17 (100%)

2nd√ Clean 2/30 (6%) 14/14 (100%)2nd √ Clean 2/30 (6%) 14/14 (100%) -

Lab C 1st √ Clean 7/102 (7%) 44/44 (100%) 18/19 (95%)

Lab D 1st Χ Contam 24/96 (25%) 48/48 (100%) 19/20(95%)Lab D 1 Χ Contam. 24/96 (25%) 48/48 (100%) 19/20(95%)

* For analytic purposes the invalid LPA results were subtracted from the denominator 6

LPA testing on Direct sputum specimen

Validation phase

7

Validation phase (1)• IRL Ahmedabad & Hyderabad & SMS Jaipur

contributed

• Pt. enrollment: Oct, 2008 – 1st week Feb, 2009

• Enrollment criteria: pre-treatment sputumEnrollment criteria: pre treatment sputum specimens sent for culture and DST

• LJ culture and DST conducted as per routine;• LJ culture and DST conducted as per routine; these results used for patient management

• Blinded LPA testing undertaken from same sputum specimens; LPA results not used for patient management

8

Validation phase (2)2 0• Target sample size: 250 specimens with valid

LPA results & LJ DST results• Specimens with discordant LPA and LJ results:

– Sequenced at JALMA & repeat DST at LRS on different system (liquid culture);

• Minimum parameter to proceed to demonstration p pphase– 95% Sensitivity to detect Rifampicin95% Sensitivity to detect Rifampicin

resistance

9

Validation phase (3)Total eligible S+ patients ota e g b e S pat e tsenrolled in study = 320

Solid media culture done320

LPA done 320320 320

Invalid LPA19 (6%)

Culture negative 51 (16%)Contaminated 13 (4%)

Culture positive256 (80%)

Specimen M.tb PCR+301 (94%)

Specimen M.tb PCR+LPA DST available

301 (94%)DST Available

256 (80%)

248 with bothLJ and LPA

DST available for analysis

RIF resistant TB141*

RIF resistant TB160

for analysis

160

*5/141 isolates had RIF mono resistance by LJ; 136 were also INH resistant10

Validation phase (4)Validation phase (4)Hyderabad Ahmedabad Jaipur TotalHyderabad Ahmedabad Jaipur Total

Total pts enrolled 123 (38%) 58 (18%) 139 (43%) 320

SMale 87 (71%) 40 (69%) 103 (75%) 230 (72%)

Female 36 (29%) 18 (31%) 35 (25%) 89 (28%)

Sex

Diagnostic 87 (71%) 58 (100%) 139 (100%) 284(89%)Others 36 (29%) - - 36 (11%)

Specimen type

Others 36 (29%) 36 (11%)

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Validation phase: LJ DST ResultsValidation phase: LJ DST Results

Hyderabad Ahmedabad Jaipur TotalNo (%) No (%) No (%) No (%)

Rif & INH Resistance 63 (63) 30 (61) 43 (40) 136 (53)Rif i & INHRif resistance & INH

susceptible 1 (1) Nil 4 (4) 5 (2)

Rif susceptible & INH 21 (21) 16 (33) 17 (16) 54 (21)presistant 21 (21) 16 (33) 17 (16) 54 (21)

Rif susceptible & INH susceptible 15 (15) 3 (6) 43 (40) 61 (24)susceptible ( ) ( ) ( ) ( )

Total DST results 100 49 107 256*

12

LPA-LJ Rif Resistance correlation (raw data before discordance resolution)

LJ

(raw data, before discordance resolution)

Resistant Sensitive TotalResistant 127 7 134

LJRIF

Resistant 127 7 134Sensitive 9 105 114

T l 136 112 248LPA

• Concordance = 94%Total 136 112 248

• Sensitivity = 93%• Specificity = 95%Specificity 95%• Total 16 discordances 13

Results from discordance resolutionSpeci.

IDLJ_RIF DST LPA_RIF

Sequencing interpretation

DST on liquid cultre at NRL

resolved

83 Resistant Sens.83 Resistant Sens.89 Resistant Sens. Sens. LPA90 Resistant Sens. Res. LJ98 Resistant Sens. Sens. Res. LJ112 Resistant Sens. Sens. Sens. LPA213 Resistant Sens. Sens. LPA270 Resistant Sens.175 R i S R R i LJ175 Resistant Sens. Res. Resistant LJ236 Resistant Sens. Sens. Resistant LJ81 Susceptib Res. Res. LPA208 Susceptib Res Res LPA208 Susceptib Res. Res. LPA235 Susceptib Res. Sens. Sens. LJ44 Susceptib Res. Res. Res. LPA70 Susceptib Res. Res. Res. LPA70 Susceptib Res. Res. Res. LPA269 Susceptib Res. Res. LPA301 Susceptib Res. Res. Res. LPA 14

LPA & LJ DST correlation: Rifampicin (2)LPA & LJ DST correlation: Rifampicin (2)• Reconciled results in view of sequencing &

DST resultsDST results– Concordance: 97%;

S iti it 96%– Sensitivity: 96%;– Specificity: 99%

Resistant Sensitive TotalRIF

LJ

Resistant 133 1 134Sensitive 6 108 114LPA

Total 139 109 24815

LPA & LJ DST correlation: Isoniazid (1)

• Concordance = 78%


• Sensitivity = 72%• Specificity = 97%• Specificity = 97%

Resistant Sensitive TotalINH

LJ

Resistant 133 2 135Sensitive 52 61 113LPA

Total 185 63 24816

LPA & LJ DST correlation: Isoniazid (2)Reconciled results in view of sequencing & DST

results


results• Concordance = 79%• Sensitivity = 72%Sensitivity 72%• Specificity = 98%

Resistant Sensitive TotalINH

LJ


T t l 185 63 248LPA

Total 185 63 24817

Spectrum of INH mutations detected by LPA i l t ith INH i t LJamong isolates with INH-resistance on LJ

n %

Kat G mutations 111 60

Inh A mutations 13 7

Inh A & Kat G mutations 9 5Inh A & Kat G mutations 9 5

No Mutation detected 52 28

Total 185 100

18

LJ DST: time to DST results and Cat IV t t t t DOTS l it *treatment at DOTS plus sites*

Time from sputum collection to DST result

Time from sputum collection to DST report

Time from sputum collection to start of

treatmentp

62 pts eligible for treat.

Median 85 90 105R 48 154 48 154 79 165Range 48-154 48-154 79-165

(N) 93 93 27

* As of 15th June, 09

*will serve as baseline for comparison with demonstration study, where patients would be initiated on treatment based on LPA result 19

LPA Testing on Culture isolates

20

LPA Testing on Culture isolates• Undertaken at Hyd & Ahd IRLs; 214 culture isolates tested;

LPA & LJ DST Correlation: Rifampicin

Resistant Sensitive TotalRIF

LJ


T l 137 77 214

LPA

Total 137 77 214

Concordance 94%Sensitivity 97%

• Raw results showed 13/214 (6%) discordancesDi d t l d t JALMA & t DST t

ySpecificity 88%

• Discordant samples sequenced at JALMA & repeat DST at LRS 21

Results from discordance resolutionLAB SPEC.

NoLJ_RIF DST LPA_RIF Sequencing

DST of liquid cultre at NRL resolved

184 Susceptib Res Resistant contam LPA184 Susceptib Res. Resistant contam LPA339 Susceptib Res. Resistant Resistant LPA538 Susceptib Res. Resistant Resistant LPA628 Susceptib Res Resistant Resistant LPA628 Susceptib Res. Resistant Resistant LPA682 Susceptib Res. Resistant Resistant LPA702 Susceptib Res. Resistant LPA731 Susceptib Res Sensitive contam LJ731 Susceptib Res. Sensitive contam LJ35 Susceptib Res. Resistant Resistant LPA41 Susceptib Res. Resistant Resistant LPA192 Resistant Sens Sensitive Sensitive LPA192 Resistant Sens. Sensitive Sensitive LPA302 Resistant Sens.144 Resistant Sens. Resistant Resistant LJ9 Resistant Sens Sensitive contam LPA9 Resistant Sens. Sensitive contam LPA

22

Reconciled results in view of sequencing & DST ltDST results

LPA & LJ DST Correlation: RifampicinLJ

Resistant Sensitive TotalResistant 141 1 142

RIFLJ


Total 143 71 214LPA

• Rif– Concordance 99%Concordance 99%– Sensitivity 99%– Specificity 99%Specificity 99%

23

SummarySummary• Mechanism for assessing lab proficiency in

conducting LPA prior to use of results developedconducting LPA prior to use of results developed and piloted

• Rif. resistance detected with high accuracy by LPA on direct sputum specimens and culture isolates– Sensitivity >95% as pre-specified in protocol

• Demonstration phase would inform RNTCP toDemonstration phase would inform RNTCP to develop specific clinical algorithms for how to use LPA in programmeLPA in programme

24

Proposed next stepsProposed next steps

• Preparation for demonstration phasePreparation for demonstration phase– Training on Study procedures (FIND)- 3rd -4th wk of

July, 09• Lab staff, concerned DTOs & DOTS plus committee

– Printing of study forms & registers (FIND)- 2 wksE t bli h t f t t t ti h i– Establishment of sputum transportation mechanisms (State, FIND)

• 3-4th wk July,09 y,

• Tentative date for the Demonstration phase: 1st

August, 09

25

Demonstration PhaseDemonstration Phase1. Duration: 6 months; August, 09- Feb, 10; g , ,

• Results to be available by April-May, 20102. Diagnostic samples from RNTCP MDR-TB

suspects for culture and DST from the project districts to be tested on LPA

3 Patient management and initiation on Cat IV3. Patient management and initiation on Cat IV treatment in these districts to be based on LPA results

4. Discordant specimen to be sent to JALMA for sequencing, repeat DST

26

Demonstration: Actions in case of LPA-LJ discordance (1)discordance (1)

LPA LJ DST Treatment Action

No valid result

Culture positive

Cat IV not started yet

•Conduct LPA from culture. Initiate Cat IV if RIF-Resistance on LPA.•Also perform LJ DST as per routine.

RIF-Resist

Culture negative/ contamin

Cat IV already started on

•Conduct DST from month-zero culture•If M0 culture also

contamin. started on basis of LPA result

negative/contaminated, then case to be reviewed by DOTS-Plus Committee for continuation of Cat IV

27

Recommended actions in case discordance (2)discordance (2)


RIF RIF Cat IV Repeat LPA immediately on primaryRIF Resist

RIF Sens.

Cat IV already started on basis of

•Repeat LPA immediately on primary diagnostic culture isolate•Repeat LJ DST from diagnostic culture isolatebasis of

LPA resultisolate. •Send DNA extract to JALMA for Sequencing and culture isolate for repeat DST on Liquid culturerepeat DST on Liquid culture

28

Recommended actions in case discordance (3)discordance (3)


RIF Sens.

RIF Resist.

No Cat IV started yet

•Initiate Cat IV treatment•Repeat LPA on primary diagnostic culture isolate•If discordance persists, send DNA extract to JALMA for Sequencing, and culture isolate for repeat DST on Liquid culture

29

ThanksThanks

30

LJResistant Sensitive Culture Contam. Total

Resistant 127 7 18 8 160

RIFLJ

Sensitive 9 105 23 4 141Invalid 5 3 10 1 19Total 141 115 51 13 320

LPA

Total 141 115 51 13 320

•Patients with LJ culture contamination with valid LPA result = 12 (4%)

•LJ culture negative patients with valid LPA result = 41 (13%)

•Pts with invalid LPA result & LJ DST results = 8 (2.5%)

31

LPA Testing on Culture isolates (1)LPA Testing on Culture isolates (1)

• Undertaken at Hyderabad & Ahmedabad IRLs• 214 culture isolates tested;

LJ

LPA & LJ DST Correlation: Rifampicin

LJ

LPA & LJ DST Correlation: Isoniazid

Resistant Sensitive Total


RIFJ

LPA

Resistant Sensitive TotalResistant 138 4 142Sensitive 38 34 72

INHLJ

LPASensitive 4 68 72Total 137 77 214


LPA Sensitive 38 34 72Total 176 38 214


LPA

• Raw results showed 13/214 (6%) discordancesDiscordant samples sequenced at JALMA &

Specificity 88% Sensitivity 78%Specificity 89%

• Discordant samples sequenced at JALMA & repeat DST at LRS 32


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Annexure-IV

MINIMUM REQUIREMENTS/SPECIFICATIONS FOR THE TUBERCULOSIS BACTERIAL CULTURE ROOMS (NEGATIVE AIR PRESSURE ROOM)

These are minimum requirements. When upgrading the existing laboratory for Setting, installation, testing, commissioning (SITC) of negative air pressure facility and further validation, take into consideration ‘on-site’ review of local conditions and available space/equipment by a certified ventilation engineer/expert and recommendations. Flexibility should be designed into the facility for fully sustainable operations, for a longer system life.

Specifications REQUIREMENT*Isolation of Containment culture room of laboratory (1) Yes Containment room Location: Ideal to have at the blind end of Lab/building or it could be anywhere with strict restricted entry/passage Yes Room sealable for decontamination (2) YesNegative air pressure Explanation: Yes Negative pressurization can be created in any room when exhausted air is approximately 20%greater than the room’s supplied air. Directional air flow is very important. Maintainingcontinuous negative air pressure in relation to the air pressure in the corridor, with a permanentlyinstalled visual monitoring mechanism (at least magnahelic gauge). Ensure that rooms are re-sealedby properly constructing windows, doors, and air-intake and exhaust ports (important for negativeair pressure maintenance). At least six air exchanges per hour (ACH) should be achieved forexisting building. New buildings or renovated facilities should achieve 12 or greater ACH.Variable Speed Drive (VSD) should be incorporated. Inward air flow direction is very importantand is determined by the differences in air pressure between adjacent areas, with air flowing fromhigh (outside) to low pressure areas (containment room). Air should flow from the hallway into thecontainment room. Air flow must be diligently monitored and documented. TB culture roomsshould be monitored for negative pressure relative to hallways and all surrounding areas. Airhandling systems connected to local or isolation rooms must be labelled with a TB warning sign.Warming signs should be located where maintenance personnel would have access to duct work,fans, or filters during maintenance or repair work. The air “balancing” should be conducted employing quality approved standard instruments(preferably by a quality certification (electronic/electrical) national/government agency approved).Certification of the measuring devices should be within the expiry dates. Negative air pressure detail:Ventilation -inward airflow HEPA filtered (0.3 micron particle size and 99.97% efficiency) air inflow. ‘Balanced’ and tested to ensure quiet, vibration free operation. Air flow ducted through ceiling with blower and filters units placed outside the building wall. Blow unit, and pre and HEPA filters housed in stainless (or powder coated) steel casings high above the ground, outside to building. Two 2" pleated anti-microbial pre-filter to protect HEPA for a longer life. Air handling systems connected to local or isolation rooms must be labelled with a TB warning sign. No re-circulation, one pass design. Yes HEPA-air exhaust to outside (3): HEPA filtered (0.3 micron and 99.97% efficiency) air exhaust to outside.Variable speed adjustment exhaust. Gel Sealed on a knife edge HEPA filter. 18"x 24" x 12" Metal Framed. Pre-filter (100mm thickness) should be 3 stageswith ASHRAE specifications e.g. 1st stage at 5-8%, 2nd stage at 30% and 3rdstage at 90% efficiency. Exhaust inside the negative pressure rooms should be protected by the Yes


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Specifications REQUIREMENT*Protection Grilles. Typical Exhaust velocity = 15-20 m/s or 3000-4000 fpm Gel pressure on a knife edge seal: prevents by-pass leakage from filter chamber to maximize filtration efficiency. Exhaust unit, and pre and HEPA filters housed in stainless (or powder coated) steel casings high above the ground outside to building. Air handling systems must be labelled with a TB warning sign. Definition of negative air pressure: Negative pressure is created by setting (or balancing) a ventilation system so that more air is mechanically exhausted from a room than is mechanically supplied. The velocity of exhaust air should be grater than inflow supply of air. Directional air flow must be established from clean areas into contaminated areas. Existing building @ at least 6 ACH (air handlings per hour), HEPA exhaust. New buildings or renovated facilities should achieve 12 or greater ACH. In a well-designed negative pressure room, air is pulled in under the door through a gap (typically half-inch high). Other than this gap, the room should be as airtight as possible to prevent air from being pulled in through cracks and gaps, such as those around windows, light fixtures, and electrical outlets. Pressure differentials across doorways must be measured using a device calibrated against a primary standard. Air supply and exhaust to be equipped with bubble tight dampers. YesSingle electrical operating switch, a single operating switch to both HEPA air in flow unit and HEPA Exhaust unit. At no point inflow Air handling unit should be allowed to run in the absence of exhaust air handling unit not working. Yes Monitors to check air pressure of containment room in comparison to outside hall-way: Monitoring methods include (a) chemical aerosols (e.g., smoke tube), (b) differential pressure-sensing devices (e.g., manometer, (suggested supplier: Dwyer Instruments, Inc, Michigan city, Indiana (USA)), a magnehelic gauge differential pressure monitoring device for continuous visual monitoring & recording; (c) and physical indicators (e.g., flutter strips) on the inside door of anteroom. Specification for Magnehelic gauge: Housing: Die cast aluminum case and bezel, with acrylic cover, Exterior finish is coated gray to withstand 168 hour salt spray corrosion test. Accuracy: +/- 2% of full scale (±3% on - 0 and ±4% on - 00 ranges), throughout range at 70°F Pressure Limits: -Should be capable for measuring the pressure differential of 0.01 inch water gauge or as appropriate to indicate negative air pressure in containment room. Overpressure: Relief plug opens at approximately 25 psig (1.72 kPa), standard gages only. Temperature Limits: 20 to 140°F.* (-6.67 to 60°C). Size: 4½ (101.6 mm) Diameter dial face. Mounting Orientation: Diaphragm in vertical position. Process Connections: 1/8½ female NPT duplicate high and low pressure taps - one pair side and one pair back. YesAudible alarms to be provided in the laboratory work area and outside laboratory zone to detect positive pressurization and air handling systems failure Yes Flooring and interior of containment room: Floor in containment room: Monolithic and slip-resistant and the continuity of seal is maintained between floor and wall. Even epoxy coated, antifungal, dust non sticky (resistant), and easy cleaning flooring is ideal. Sealing in containment room: All wall air leakage spaces, and penetrations into the containment room, are sealed with smooth finish or non-shrinking sealant to prevent air leakage to facilitate decontamination. Interior surfaces, coatings and finishes in containment room: should be smooth, continuous, impermeable to liquids, detergents, disinfectants and Yes


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Specifications REQUIREMENT*decontamination gases (such as formaldehyde) normally used in containment lab. Fire Exit door: single-door, should lead to outside anteroom or air-lock (if is provided) Pass Boxes: All pass-through cabinets should be made of stainless steel with mechanical interlocks with short wavelength UV light for decontamination of the pass box. Dimension: Min 60 cm x 60cm size outer to outer. Ideally, placed through anteroom room into containment room. Yes Access through Anteroom Yes Double-door entry to containment room (i.e., leading to containment room through anteroom). Each door is single plane; preferably see through, half-glass, half aluminum with hydraulic door closures firmly fixed; or a self closing device). Need for self closing device such as magnetic door-lock (air interlock), providing “Air-lock” is preferred. Yes Anteroom with hand-wash sink with "hands-free" capability or at least elbow operated. DesirableAutoclave (Sterilizer) Horizontal double chamber type- on site (accessible to the infectious-waste safe disposal from containment room without carrying thorough hall-ways or through-fare passage ways). (5) Yes Biological safety cabinets (BSC) CDC class II, ducted outside (4) YesPersonnel safety Monitoring Capacity YesBack-up electrical supply (Power Generator, Should not be placed attached to the building, should be placed with min 3 meters distance, to avoid unnecessary vibrations). For power back-up UPS as well as silent Diesel Generator set of sufficient capacity should be provided, indicating power requirement of the laboratory with break-up. UPS should be of minimum 30 minutes back-up and Diesel Generator (DG) set should automatically start operating within 5 minutes. Yes Principle Standards: The negative air pressure facility should be designed and complying to the Laboratory Bio-safety Manual-WHO, GENEVA – 3rd edition (2004) Reference standards: Bio-safety in Microbiological and Biomedical Laboratories – 5th Edition (2007).Other international guidelines and standards such as ASHRAE, NIH, EU, EN in this regard. Regarding Civil, Electrical and Mechanical latest relevant International Standards shall be applicable. Yes Validation: Should include the following, at the minimum: 1. Negative air-pressure in different sections of the laboratory is to be

measured. There should be provision of alternative equipment other than theones fitted in the control panel or the computer for measurement.Measurements are also to be taken both in power-trip power back-up mode.

2. Validation of all engineering controls against respective primary standards. 3. Performance tests: Room differential pressure test verification, particle test

for cleanliness, air pattern smoke test, Air velocity test, HEPA filter leak testand Wall monitors and panels check for performance. All validated againststandards.

4. Air-locks should be tested for compliance. 5. Validation of correct placement of biological safety cabinets with respect to Yes


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Specifications REQUIREMENT*supply and exhaust diffusers, doors and traffic patterns.

6. Leakage in ducts, plenums and doors should be checked for compliance. 7. Bio-safety cabinets should be validated and certified. 8. The training should be provided to responsible officers at the lab for day-to-

day operations and maintenance of each component and the equipmentsprovided. A manual of operations and maintenance should be posted andprovided to labs.

(1) The containment laboratory must be separated from the areas that are open to unrestricted traffic flow within the building. Additional separation may be achieved by placing the laboratory at the blind end of a corridor, or constructing a partition and door or access through an anteroom (e.g. a double-door entry), describing a specific area designed to maintain the pressure differential between the laboratory and its adjacent space. The anteroom should have facilities for separating clean and dirty clothing. It is strongly recommended that the BSL level II lab should lead to ‘negative air pressure containment’ facility/room. (2) Windows must be closed, sealed and break-resistant. Room should be sealed just enough allowing decontamination by gaseous methods, practicable. (3) The air from the containment laboratory is not re-circulated to other areas within the building. Exhaust air from the laboratory is discharged to the outside of the building, should be dispersed away from occupied buildings, hospital wards, patient reception rooms, doctor consultation rooms, and other air intakes. (4) All bio-safety cabinets must be vented to the outside, either with hard duct or with thimble connection. (5) An autoclave for the decontamination of contaminated waste material should be available in the containment laboratory. If infectious waste has to be removed from the containment laboratory for decontamination and disposal, it must be transported in sealed, unbreakable and leak-proof containers according to national or international regulations, as appropriate. (6) Important factors to define bio-safe containment rooms are 1) physical requirements, directional air-flow, as mentioned above and sustained maintenance of the equipment and installation, 2) adherence to BSL3 practice using protocols minimizing aerosol production and spill, 3) adequate training of staff.


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Annexure-V Guidance on laboratory design for molecular line probe assay facilities: Work flow in molecular diagnostic lab is: (a) Sample preparation (DNA extraction) (b) preparation of regents for setting up PCR (master mix or pre-mix preparation) (c) PCR amplification, and hybridisation (d) post-PCR analysis of results. Preventing amplicon contamination is single most crucial need of the facility reducing the risk of cross-contamination and false results. This is achieved by physically separating the source of amplicons’ (e.g., post-PCR) activities from the Pre-PCR activities. The facility is placed in close proximity to the C&DST facility. Ideal arrangement of the molecular diagnostic facility is to have separate rooms. However, due to space constraints, separate rooms for each step of work-flow might not possible, and renovation of existing facility is required. Two to three separate rooms are suggested. Room are fairly small in size. Alternately, a big room could be partitioned, appropriately. All efforts are ensured to prevent dust by restricting the access to facility, sealing off wall openings to exterior and meticulously following the cleaning procedures for rooms & work-benches. Dedicated supplies (such as micropipettes, sterile disposable plugged pipette tips, microcentrifuge tubes, reagents, distilled water, cleaning solutions and floor cleaning mops etc.,) need to be established in each room, separately. An almirah underneath benches to keep dedicated pipette sets for all three rooms is advisable. Facility detail (fig 1):

1. Sample preparation (DNA extraction room): No separate room is required for this activity. It is performed in the culture and DST room, itself, inside the biological safety cabinet. DNA is extracted directly from the processed sputum sediment by heating and/ sonication. A dedicated work-bench and using the disposable gloves (with frequent changes) would help in avoiding sample-to-sample target DNA contamination. Thoroughly clean the work surfaces with 10% bleach followed by isopropyl alcohol and/or ethanol before setting the work area.

2. Pre-mix or master-mix room: This work area is used for making master-mix for setting up PCR (e.g., dNTPs, 10X buffer, distilled water, taq DNA polymerase). Double door entry is preferred. A clean-work bench need to be available in this work area. A small refrigerator and/ a -20ºc freezer (small but double sleeve), and a quick-spin need to be available within the reach.

3. Amplification room: This work area is meant for the PCR amplification of target DNA.

4. Hybridisation room: This work area is meant for handling the amplified DNA for hybridisation.

(Wherever extra space is not available, the amplification and hybridisation work is carried out on separate work-benches with in a single room (fig 2). One work bench/area is meant for PCR amplification. The second work bench/area is meant for those activities which involve the handling of amplified DNA- i.e., hybridisation with Line probe assay strips).


Fig1: Suggested floor sketch 1 (6x7 meters)

Fig 2: Suggested floor sketch 2 (6x7 meters)

(1 x 3 mt)

(3 x 3 mt) (3 x 4 mt) (3 x 4 mt)

(3 x 3 mt)

(1 x 3 mt)

(4 x 3 mt)

(1.8 x 7 mt)

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