Development and validation of quantitative PCR assays for HIV-associated cryptococcal meningitis in sub-Saharan Africa: a diagnostic accuracy study

Tshepiso Mbangiwa; Aude Sturny-Leclère; Kwana Lechiile; Cheusisime Kajanga; Timothée Boyer-Chammard; Jennifer C. Hoving; Tshepo Leeme; Melanie Moyo; Nabila Youssouf; David S. Lawrence; Henry Mwandumba; Mosepele Mosepele; Thomas S. Harrison; Joseph N. Jarvis; Olivier Lortholary; Alexandre Alanio; J. Goodall; N. Mawoko; J. Milburn; R. Mmipi; C. Muthoga; P. Ponatshego; I. Rulaganyang; K. Seatla; N. Tlhako; K. Tsholo; S. April; A. Bekiswa; L. Boloko; H. Bookholane; T. Crede; L. Davids; R. Goliath; S. Hlungulu; R. Hoffman; H. Kyepa; N. Masina; D. Maughan; T. Mnguni; S. Moosa; T. Morar; M. Mpalali; J. Naude; I. Oliphant; S. Sayed; L. Sebesho; M. Shey; L. Swanepoel; M. Chasweka; W. Chimang'anga

doi:10.1016/s2666-5247(23)00362-2

Development and validation of quantitative PCR assays for HIV-associated cryptococcal meningitis in sub-Saharan Africa: a diagnostic accuracy study

Tshepiso Mbangiwa, Aude Sturny-Leclère, Kwana Lechiile, Cheusisime Kajanga, Timothée Boyer-Chammard, Jennifer C. Hoving, Tshepo Leeme, Melanie Moyo, Nabila Youssouf, David S. Lawrence, Henry Mwandumba, Mosepele Mosepele, Thomas S. Harrison, Joseph N. Jarvis, Olivier Lortholary, Alexandre Alanio, J. Goodall, N. Mawoko, J. Milburn, R. MmipiC. Muthoga, P. Ponatshego, I. Rulaganyang, K. Seatla, N. Tlhako, K. Tsholo, S. April, A. Bekiswa, L. Boloko, H. Bookholane, T. Crede, L. Davids, R. Goliath, S. Hlungulu, R. Hoffman, H. Kyepa, N. Masina, D. Maughan, T. Mnguni, S. Moosa, T. Morar, M. Mpalali, J. Naude, I. Oliphant, S. Sayed, L. Sebesho, M. Shey, L. Swanepoel, M. Chasweka, W. Chimang'anga

Clinical Sciences

Research output: Contribution to journal › Article › peer-review

11 Citations (Scopus)

Abstract

Background

HIV-associated cryptococcal meningitis is the second leading cause of AIDS-related deaths, with a 10-week mortality rate of 25–30%. Fungal load assessed by colony-forming unit (CFU) counts is used as a prognostic marker and to monitor response to treatment in research studies. PCR-based assessment of fungal load could be quicker and less labour-intensive. We sought to design, optimise, and validate quantitative PCR (qPCR) assays for the detection, identification, and quantification of Cryptococcus infections in patients with cryptococcal meningitis in sub-Saharan Africa.

Methods

We developed and validated species-specific qPCR assays based on DNA amplification of QSP1 (QSP1A specific to Cryptococcus neoformans, QSP1B/C specific to Cryptococcus deneoformans, and QSP1D specific to Cryptococcus gattii species) and a pan-Cryptococcus assay based on a multicopy 28S rRNA gene. This was a longitudinal study that validated the designed assays on cerebrospinal fluid (CSF) of 209 patients with cryptococcal meningitis at baseline (day 0) and during anti-fungal therapy (day 7 and day 14), from the AMBITION-cm trial in Botswana and Malawi (2018–21). Eligible patients were aged 18 years or older and presenting with a first case of cryptococcal meningitis.

Findings

When compared with quantitative cryptococcal culture as the reference, the sensitivity of the 28S rRNA was 98·2% (95% CI 95·1–99·5) and of the QSP1 assay was 90·4% (85·2–94·0) in CSF at day 0. Quantification of the fungal load with QSP1 and 28S rRNA qPCR correlated with quantitative cryptococcal culture (R2=0·73 and R2=0·78, respectively). Both Botswana and Malawi had a predominant C neoformans prevalence of 67% (95% CI 55–75) and 68% (57–73), respectively, and lower C gattii rates of 21% (14–31) and 8% (4–14), respectively. We identified ten patients that, after 14 days of treatment, harboured viable but non-culturable yeasts based on QSP1 RNA detection (without any positive CFU in CSF culture).

Interpretation

QSP1 and 28S rRNA assays are useful in identifying Cryptococcus species. qPCR results correlate well with baseline quantitative cryptococcal culture and show a similar decline in fungal load during induction therapy. These assays could be a faster alternative to quantitative cryptococcal culture to determine fungal load clearance. The clinical implications of the possible detection of viable but non-culturable cells in CSF during induction therapy remain unclear.

Original language	English
Pages (from-to)	e261-e271
Journal	The Lancet Microbe
Volume	5
Issue number	3
DOIs	https://doi.org/10.1016/s2666-5247(23)00362-2
Publication status	Published - 8 Feb 2024

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Mbangiwa, T., Sturny-Leclère, A., Lechiile, K., Kajanga, C., Boyer-Chammard, T., Hoving, J. C., Leeme, T., Moyo, M., Youssouf, N., Lawrence, D. S., Mwandumba, H., Mosepele, M., Harrison, T. S., Jarvis, J. N., Lortholary, O., Alanio, A., Goodall, J., Mawoko, N., Milburn, J., ... Chimang'anga, W. (2024). Development and validation of quantitative PCR assays for HIV-associated cryptococcal meningitis in sub-Saharan Africa: a diagnostic accuracy study. The Lancet Microbe, 5(3), e261-e271. https://doi.org/10.1016/s2666-5247(23)00362-2

@article{4e796e3d53fb4d38ad0153f8ae82ff3f,

title = "Development and validation of quantitative PCR assays for HIV-associated cryptococcal meningitis in sub-Saharan Africa: a diagnostic accuracy study",

abstract = "BackgroundHIV-associated cryptococcal meningitis is the second leading cause of AIDS-related deaths, with a 10-week mortality rate of 25–30\%. Fungal load assessed by colony-forming unit (CFU) counts is used as a prognostic marker and to monitor response to treatment in research studies. PCR-based assessment of fungal load could be quicker and less labour-intensive. We sought to design, optimise, and validate quantitative PCR (qPCR) assays for the detection, identification, and quantification of Cryptococcus infections in patients with cryptococcal meningitis in sub-Saharan Africa.MethodsWe developed and validated species-specific qPCR assays based on DNA amplification of QSP1 (QSP1A specific to Cryptococcus neoformans, QSP1B/C specific to Cryptococcus deneoformans, and QSP1D specific to Cryptococcus gattii species) and a pan-Cryptococcus assay based on a multicopy 28S rRNA gene. This was a longitudinal study that validated the designed assays on cerebrospinal fluid (CSF) of 209 patients with cryptococcal meningitis at baseline (day 0) and during anti-fungal therapy (day 7 and day 14), from the AMBITION-cm trial in Botswana and Malawi (2018–21). Eligible patients were aged 18 years or older and presenting with a first case of cryptococcal meningitis.FindingsWhen compared with quantitative cryptococcal culture as the reference, the sensitivity of the 28S rRNA was 98·2\% (95\% CI 95·1–99·5) and of the QSP1 assay was 90·4\% (85·2–94·0) in CSF at day 0. Quantification of the fungal load with QSP1 and 28S rRNA qPCR correlated with quantitative cryptococcal culture (R2=0·73 and R2=0·78, respectively). Both Botswana and Malawi had a predominant C neoformans prevalence of 67\% (95\% CI 55–75) and 68\% (57–73), respectively, and lower C gattii rates of 21\% (14–31) and 8\% (4–14), respectively. We identified ten patients that, after 14 days of treatment, harboured viable but non-culturable yeasts based on QSP1 RNA detection (without any positive CFU in CSF culture).InterpretationQSP1 and 28S rRNA assays are useful in identifying Cryptococcus species. qPCR results correlate well with baseline quantitative cryptococcal culture and show a similar decline in fungal load during induction therapy. These assays could be a faster alternative to quantitative cryptococcal culture to determine fungal load clearance. The clinical implications of the possible detection of viable but non-culturable cells in CSF during induction therapy remain unclear.",

author = "Tshepiso Mbangiwa and Aude Sturny-Lecl{\`e}re and Kwana Lechiile and Cheusisime Kajanga and Timoth{\'e}e Boyer-Chammard and Hoving, \{Jennifer C.\} and Tshepo Leeme and Melanie Moyo and Nabila Youssouf and Lawrence, \{David S.\} and Henry Mwandumba and Mosepele Mosepele and Harrison, \{Thomas S.\} and Jarvis, \{Joseph N.\} and Olivier Lortholary and Alexandre Alanio and J. Goodall and N. Mawoko and J. Milburn and R. Mmipi and C. Muthoga and P. Ponatshego and I. Rulaganyang and K. Seatla and N. Tlhako and K. Tsholo and S. April and A. Bekiswa and L. Boloko and H. Bookholane and T. Crede and L. Davids and R. Goliath and S. Hlungulu and R. Hoffman and H. Kyepa and N. Masina and D. Maughan and T. Mnguni and S. Moosa and T. Morar and M. Mpalali and J. Naude and I. Oliphant and S. Sayed and L. Sebesho and M. Shey and L. Swanepoel and M. Chasweka and W. Chimang'anga",

year = "2024",

month = feb,

day = "8",

doi = "10.1016/s2666-5247(23)00362-2",

language = "English",

volume = "5",

pages = "e261--e271",

journal = "The Lancet Microbe",

issn = "2666-5247",

publisher = "Elsevier Ltd",

number = "3",

}

Mbangiwa, T, Sturny-Leclère, A, Lechiile, K, Kajanga, C, Boyer-Chammard, T, Hoving, JC, Leeme, T, Moyo, M, Youssouf, N, Lawrence, DS, Mwandumba, H, Mosepele, M, Harrison, TS, Jarvis, JN, Lortholary, O, Alanio, A, Goodall, J, Mawoko, N, Milburn, J, Mmipi, R, Muthoga, C, Ponatshego, P, Rulaganyang, I, Seatla, K, Tlhako, N, Tsholo, K, April, S, Bekiswa, A, Boloko, L, Bookholane, H, Crede, T, Davids, L, Goliath, R, Hlungulu, S, Hoffman, R, Kyepa, H, Masina, N, Maughan, D, Mnguni, T, Moosa, S, Morar, T, Mpalali, M, Naude, J, Oliphant, I, Sayed, S, Sebesho, L, Shey, M, Swanepoel, L, Chasweka, M & Chimang'anga, W 2024, 'Development and validation of quantitative PCR assays for HIV-associated cryptococcal meningitis in sub-Saharan Africa: a diagnostic accuracy study', The Lancet Microbe, vol. 5, no. 3, pp. e261-e271. https://doi.org/10.1016/s2666-5247(23)00362-2

TY - JOUR

T1 - Development and validation of quantitative PCR assays for HIV-associated cryptococcal meningitis in sub-Saharan Africa: a diagnostic accuracy study

AU - Mbangiwa, Tshepiso

AU - Sturny-Leclère, Aude

AU - Lechiile, Kwana

AU - Kajanga, Cheusisime

AU - Boyer-Chammard, Timothée

AU - Hoving, Jennifer C.

AU - Leeme, Tshepo

AU - Moyo, Melanie

AU - Youssouf, Nabila

AU - Lawrence, David S.

AU - Mwandumba, Henry

AU - Mosepele, Mosepele

AU - Harrison, Thomas S.

AU - Jarvis, Joseph N.

AU - Lortholary, Olivier

AU - Alanio, Alexandre

AU - Goodall, J.

AU - Mawoko, N.

AU - Milburn, J.

AU - Mmipi, R.

AU - Muthoga, C.

AU - Ponatshego, P.

AU - Rulaganyang, I.

AU - Seatla, K.

AU - Tlhako, N.

AU - Tsholo, K.

AU - April, S.

AU - Bekiswa, A.

AU - Boloko, L.

AU - Bookholane, H.

AU - Crede, T.

AU - Davids, L.

AU - Goliath, R.

AU - Hlungulu, S.

AU - Hoffman, R.

AU - Kyepa, H.

AU - Masina, N.

AU - Maughan, D.

AU - Mnguni, T.

AU - Moosa, S.

AU - Morar, T.

AU - Mpalali, M.

AU - Naude, J.

AU - Oliphant, I.

AU - Sayed, S.

AU - Sebesho, L.

AU - Shey, M.

AU - Swanepoel, L.

AU - Chasweka, M.

AU - Chimang'anga, W.

PY - 2024/2/8

Y1 - 2024/2/8

N2 - BackgroundHIV-associated cryptococcal meningitis is the second leading cause of AIDS-related deaths, with a 10-week mortality rate of 25–30%. Fungal load assessed by colony-forming unit (CFU) counts is used as a prognostic marker and to monitor response to treatment in research studies. PCR-based assessment of fungal load could be quicker and less labour-intensive. We sought to design, optimise, and validate quantitative PCR (qPCR) assays for the detection, identification, and quantification of Cryptococcus infections in patients with cryptococcal meningitis in sub-Saharan Africa.MethodsWe developed and validated species-specific qPCR assays based on DNA amplification of QSP1 (QSP1A specific to Cryptococcus neoformans, QSP1B/C specific to Cryptococcus deneoformans, and QSP1D specific to Cryptococcus gattii species) and a pan-Cryptococcus assay based on a multicopy 28S rRNA gene. This was a longitudinal study that validated the designed assays on cerebrospinal fluid (CSF) of 209 patients with cryptococcal meningitis at baseline (day 0) and during anti-fungal therapy (day 7 and day 14), from the AMBITION-cm trial in Botswana and Malawi (2018–21). Eligible patients were aged 18 years or older and presenting with a first case of cryptococcal meningitis.FindingsWhen compared with quantitative cryptococcal culture as the reference, the sensitivity of the 28S rRNA was 98·2% (95% CI 95·1–99·5) and of the QSP1 assay was 90·4% (85·2–94·0) in CSF at day 0. Quantification of the fungal load with QSP1 and 28S rRNA qPCR correlated with quantitative cryptococcal culture (R2=0·73 and R2=0·78, respectively). Both Botswana and Malawi had a predominant C neoformans prevalence of 67% (95% CI 55–75) and 68% (57–73), respectively, and lower C gattii rates of 21% (14–31) and 8% (4–14), respectively. We identified ten patients that, after 14 days of treatment, harboured viable but non-culturable yeasts based on QSP1 RNA detection (without any positive CFU in CSF culture).InterpretationQSP1 and 28S rRNA assays are useful in identifying Cryptococcus species. qPCR results correlate well with baseline quantitative cryptococcal culture and show a similar decline in fungal load during induction therapy. These assays could be a faster alternative to quantitative cryptococcal culture to determine fungal load clearance. The clinical implications of the possible detection of viable but non-culturable cells in CSF during induction therapy remain unclear.

AB - BackgroundHIV-associated cryptococcal meningitis is the second leading cause of AIDS-related deaths, with a 10-week mortality rate of 25–30%. Fungal load assessed by colony-forming unit (CFU) counts is used as a prognostic marker and to monitor response to treatment in research studies. PCR-based assessment of fungal load could be quicker and less labour-intensive. We sought to design, optimise, and validate quantitative PCR (qPCR) assays for the detection, identification, and quantification of Cryptococcus infections in patients with cryptococcal meningitis in sub-Saharan Africa.MethodsWe developed and validated species-specific qPCR assays based on DNA amplification of QSP1 (QSP1A specific to Cryptococcus neoformans, QSP1B/C specific to Cryptococcus deneoformans, and QSP1D specific to Cryptococcus gattii species) and a pan-Cryptococcus assay based on a multicopy 28S rRNA gene. This was a longitudinal study that validated the designed assays on cerebrospinal fluid (CSF) of 209 patients with cryptococcal meningitis at baseline (day 0) and during anti-fungal therapy (day 7 and day 14), from the AMBITION-cm trial in Botswana and Malawi (2018–21). Eligible patients were aged 18 years or older and presenting with a first case of cryptococcal meningitis.FindingsWhen compared with quantitative cryptococcal culture as the reference, the sensitivity of the 28S rRNA was 98·2% (95% CI 95·1–99·5) and of the QSP1 assay was 90·4% (85·2–94·0) in CSF at day 0. Quantification of the fungal load with QSP1 and 28S rRNA qPCR correlated with quantitative cryptococcal culture (R2=0·73 and R2=0·78, respectively). Both Botswana and Malawi had a predominant C neoformans prevalence of 67% (95% CI 55–75) and 68% (57–73), respectively, and lower C gattii rates of 21% (14–31) and 8% (4–14), respectively. We identified ten patients that, after 14 days of treatment, harboured viable but non-culturable yeasts based on QSP1 RNA detection (without any positive CFU in CSF culture).InterpretationQSP1 and 28S rRNA assays are useful in identifying Cryptococcus species. qPCR results correlate well with baseline quantitative cryptococcal culture and show a similar decline in fungal load during induction therapy. These assays could be a faster alternative to quantitative cryptococcal culture to determine fungal load clearance. The clinical implications of the possible detection of viable but non-culturable cells in CSF during induction therapy remain unclear.

U2 - 10.1016/s2666-5247(23)00362-2

DO - 10.1016/s2666-5247(23)00362-2

M3 - Article

SN - 2666-5247

VL - 5

SP - e261-e271

JO - The Lancet Microbe

JF - The Lancet Microbe

IS - 3

ER -

Development and validation of quantitative PCR assays for HIV-associated cryptococcal meningitis in sub-Saharan Africa: a diagnostic accuracy study

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