Delivering on the Antimicrobial Resistance (AMR) agenda is not possible without improving fungal diagnostic capabilities

David W. Denning1,2, David. S. Perlin2,3, Eavan G. Muldoon1, Arnaldo Lopes Colombo2,4, Arunaloke Chakrabarti2,5, Malcolm D. Richardson2.6 and Tania C. Sorrell2,7

1 The National Aspergillosis Centre, University Hospital of South Manchester, The University of Manchester, Manchester Academic Health Science Centre, Manchester, UK

2 Global Action Fund for Fungal Infections, Geneva, Switzerland;

3 Public Health Research Institute, Rutgers Biomedical and Health Sciences, Newark, NJ, USA;

4 Division of Infectious Diseases, Universidade Federal de São Paulo, São Paulo, Brazil.

5 Center of Advance Research in Medical Mycology, Postgraduate Institute of Medical Education & Research, Chandigarh 160012, India

6 Mycology Reference Centre, Manchester; Manchester Academic Health Science Centre; University Hospital of South Manchester, Manchester and International Society for Human and Animal Mycology

7Marie Bashir Institute for Infectious Diseases & Biosecurity, University of Sydney, Australia.

*Correspondence:

Professor David Denning

National Aspergillosis Centre, Education and Research Centre, University Hospital of

South Manchester, Southmoor Road, Manchester M23 9LT

Tel.: +44 161 291 5811

E-mail: ddenning@manchester.ac.uk

One line summary: Widespread use of accurate and sensitive fungal diagnostic testing will reduce unnecessary antibacterial and antifungal therapy

Key words: Aspergillus, Candida, Pneumocystis, Histoplasma, Cryptococcus, resistance

Word count: 2196

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ABSTRACT

Antimicrobial resistance (AMR) is a major public health concern largely arising from

the excess use of antibacterial and antifungal drugs. Here we argue that the lack of

routine fungal disease diagnostic testing exacerbates the problem of antimicrobial

empiricism, both antibiotic and antifungal. We cite 4 common clinical situations that

are illustrative. 1. Accurate diagnosis of fungal sepsis in the hospital and the

Intensive Care Unit to reduce the use of broad-spectrum antibacterial therapy

administered inappropriately to patients with invasive candidiasis. 2. Failure to

diagnose smear-negative pulmonary ‘tuberculosis’ as chronic pulmonary

aspergillosis. 3. Mis-diagnosing ‘fungal asthma’ that results in unnecessary courses of

antibiotics instead of antifungals and missing life-threatening invasive aspergillosis in

COPD patients. 4. Both over- and under-treatment of Pneumocystis jirovecii

pneumonia in HIV positive patients. All communities should have access to non-

culture fungal diagnostics, which will have a substantial benefit for clinical outcome,

antimicrobial stewardship and AMR control.

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Antimicrobial resistance (AMR) is a significant public health concern, and a major

threat to modern medicine (1). It is estimated that in the United States antibiotic

resistant infections are associated with 23,000 deaths per year (2), and that excess

healthcare costs are in the region of US $20-25 billion (3). Minimizing AMR has been

the focus of accelerating efforts with multi-pronged approaches tailored to individual

countries and healthcare settings. Even if the difficult task of developing new

antimicrobials is successful, current efforts aimed at reducing the development of

resistance will need to be maintained to protect these novel compounds.

A central tenet of controlling AMR is antibiotic stewardship, which seeks to limit

inappropriate antibiotic usage either by avoiding unnecessary prescribing or

discontinuing antibiotic therapy immediately it is not required. Within the context of

stewardship programs, inadequate attention has been paid to fungal infection as the

cause of antibacterial treatment failure. Furthermore the importance of accurate and

timely diagnosis of fungal infection in defeating AMR has been starkly absent from

policy discussions (4). Accurate diagnosis or exclusion of fungal infection impacts

substantially on antimicrobial usage and our ability to limit bacterial AMR.

Generally, physicians prescribe most antimicrobials empirically as very few

infections are microbiologically diagnosed in real time. Further, most infections are

never microbiologically documented, which matters little if the patient improves and

infection resolves, but leads to additional empirical antimicrobial use if the patient

deteriorates (5). Fungal infections are often undiagnosed and untreated, and they are a

frequent fatal complication (superinfection) of numerous diseases (cancer, liver,

respiratory and/or renal failure, sepsis, AIDS etc) that contribute to inappropriate

antibiotic usage. This must stop.

We provide four specific clinical examples where we argue for greater application

of existing fungal diagnostics and improved overall fungal diagnostic capability in

line with the recently issued ’95-95 by 2025’ Roadmap from the Global Action Fund

for Fungal Infections (6).

Accurate diagnosis of fungal sepsis in hospital and Intensive Care Unit (ICU)

Hospitalized patients, especially those in the ICU, are often inappropriately placed on

broad-spectrum antibiotics because fungal diseases involving Candida spp are not

diagnosed. Bloodstream infection and invasive candidiasis are substantially more

common than appreciated. Multiple factors are likely to be responsible, including

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unrestrained antibiotic use, indwelling devices, increasing populations of

immunocompromised patients, increased renal support and others. Bloodstream

infections with Candida spp. have a population incidence of 1.2-26/100,000 in

multiple studies, with the highest rates in middle- and high-income countries, notably

the USA (7,8). Most (93%) of episodes are healthcare-associated (~80% as inpatients)

and 7% are community acquired (9). In Brazil, with a population of 194 million,

Candida bloodstream infections are seen in 14.9/100,000 population, or 29,000

people each year (10), based on prospective data obtained 10 years ago from 11

medical centres (11). A recent prospective study from 27 ICUs in India found a mean

incidence of 6.51 cases of ICU-acquired candidemia per 1,000 ICU admissions with

a 35-75% mortality (12). An estimated 14.3 million patients are admitted to ICUs in

India each year. Undoubtedly this high rate underestimates the problem, as blood

culture is only ~40% sensitive for invasive candidiasis (including intra-abdominal

candidiasis), with fluconazole and echinocandins substantially reducing the yield from

blood culture (13-15). It is likely therefore that the actual burden and death rate in

ICU exceeds 200,000 patients and ~100,000 deaths. If, as is found in other countries,

Candida bloodstream infection (and presumably invasive candidiasis) outside ICUs is

at least twice as common as in ICUs, over 600,000 patients are estimated to have

invasive candidiasis in India each year, with ~300,000 deaths, assuming patients are

treated, and many more if not.

In addition, a high prevalence of candidemia has been reported in children and

neonates in India and Latin America. Central nervous system involvement is quite

common in premature infants leading to a high rate of neurological sequelae (16).

While invasive candidiasis is strongly associated with prior bacterial infection and

anti-bacterial therapy, inappropriate escalation and combination antibacterial therapy

will typically have been administered to patients with invasive candidiasis. In one

large study of 444 patients with Candida bloodstream infections, 81% were exposed

to multiple antibacterial drugs, either concomitantly or sequentially (17) and in the

ICU study from India described above, 95% were already on antibiotics, usually two

or more (12). Early therapy of Candida bloodstream infection greatly improves

patient outcomes as shown in several observational studies, and even better if

‘correct’ therapy is given immediately (18).

Once Candida sepsis is confirmed, antibacterial agents can usually be stopped and,

if ruled out, empirical antifungal therapy can be stopped. Inflammation without

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infection requires no antimicrobial therapy. Three well-validated diagnostic tools, two

configured for ruling out a diagnosis of invasive candidiasis, are now available - the

1,3 beta-D-glucan assay (19), the Candida albicans germ tube antibody test (CAGTA)

(serum samples) (20) and a newly FDA approved nonculture-based molecular assay

which is substantially more sensitive than blood culture and useful for making the

diagnosis Candida (EDTA blood) (21). In patients without invasive candidiasis,

antimicrobial stewardship programs based on such diagnostics have successfully

curtailed the use of antifungal therapy in the ICU without worsening patient outcomes

(18,22). The economics depend on the cost of diagnostic reagents and testing,

antifungal costs and incidence of infection (23). Overuse of antifungal agents is very

costly and can promote antifungal resistance, may carry toxicity and the potential for

numerous detrimental drug interactions (18). Modeling will be required to determine

the magnitude of the impact on antibacterial prescribing. What is not in dispute is that

patient outcomes for those with fungal infection will improve. Antimicrobial

stewardship programs will be even more effective if these diagnostic tools, with their

rapid turnaround times, are readily available (24). Widespread implementation of

rapid non-culture Candida diagnostics will greatly improve prescribing practices for

the complex hospital patient.

Mis-diagnosis of smear-negative pulmonary ‘tuberculosis’ as tuberculosis

Smear-negative pulmonary tuberculosis (TB) is a problematic area for clinicians

and policy makers. Post-tuberculous sequelae are common, poorly studied and may be

mistaken for active, recurrent TB (25), and it is apparent that an important under-

recognised issue for these patients is chronic pulmonary aspergillosis (CPA), which

can mimic TB presentation. Among 544 patients previously treated for TB, with a

residual cavity, in the UK, 34% developed precipitating antibodies to Aspergillus

fumigatus of whom 63% developed aspergilloma within 2 years (a late stage of

chronic pulmonary aspergillosis) and 42% of these patients developed hemoptysis

(26). Prospective studies of CPA (after treatment for TB) are otherwise lacking, but

conservatively an assumed rate of ~10% is likely, with ~1.2 million individuals

affected (26).

Culture of Mycobacterium tuberculosis from smear-negative patients is slow and

may be falsely negative. The use of new, highly sensitive, DNA detection assays (e.g.

Xpert MTB/RIF) directly on respiratory specimens has transformed the rapidity of

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detecting positive results, but there remains a considerable proportion of unwell,

smear negative, PCR-negative patients. Some of these are patients who have

‘relapsed’ following anti-tuberculous therapy have CPA. Those with ‘smear-negative

TB’ and HIV infection, have a higher mortality than smear positive patients (27),

probably because many do not have TB at all. It is increasingly being recognized that

many of these patients are chronically infected with Aspergillus spp. resulting in CPA

that is largely undiagnosed and untreated.

Weight loss, worsening cough, chest pains, dyspnea and fatigue are common to TB

and CPA, and the radiological abnormalities are similar (Figure 1). In studies from the

UK, Brazil, Korea, Iran and India, the frequency of elevated Aspergillus serum

antibody after TB varied upwards from 20% (28,29. Aspergillus antibody detection is

the key diagnostic test for CPA, and performs well (96-97% sensitivity, 92-98%

specificity (30,31), compared with controls. Given that CPA is a common sequel to

TB and has a 5-year mortality of 75-80%, it needs to be sought actively using

antibody testing (32). Sera from almost all patients with ‘recurrent TB’ in Iran were

Aspergillus antibody positive (33). Pneumocystis jirovecii DNA was identified in the

sputum of up to 7% of patients with smear-negative TB in Brazil and 6.7% in Uganda,

providing an alternative, potentially treatable diagnosis, and there are others such as

histoplasmosis (Figure 2) and coccidioidomycosis (5).

Empirical anti-tuberculous therapy is unnecessary in patients with either chronic

pulmonary aspergillosis or other fungal infections, as it is ineffective and exposes the

patient to potential toxicity. Yet, it remains the default approach for most complicated

TB patients. When such treatment fails, as it usually does, there is a risk that patients

are assumed to have multidrug resistant (MDR) TB, and the inappropriate substitution

of second and third line anti-tuberculous agents adds to potential toxicities and

healthcare costs. The size of the problem is substantial with the WHO reporting

2,755,870 patients with smear negative TB in 2013 (34). Aspergillus antibody

detection should be widely available and integrated into TB control programs with

CPA as post-TB sequelae emphasised. All symptomatically recurrent TB cases should

be screened for Aspergillus antibody.

Fungal exacerbations of asthma and chronic obstructive airways disease (COPD)

It is common practice in the community to treat asthma and exacerbations of

COPD with antibiotics and corticosteroids, although guidelines caution against

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unnecessary use, especially in COPD patients (35). Most patients respond, even if the

exacerbation is virus-induced. Patients with moderate and severe COPD are

frequently admitted to hospital and have an in-hospital mortality of 6%. The majority

patients (>80%) are treated with antibiotics (36), although multiple guidelines advise

against this in the absence of purulent sputum and pulmonary infiltrates. It is now well

established that Aspergillus airways colonization (or infection) is strongly associated

with exacerbations (37). Two studies have addressed the frequency of invasive

aspergillosis in these patients - with a 1.3% rate in Spain (38) and a 3.9% rate in

southern China (39), based on sputum cultures revealing Aspergillus spp (which likely

under-estimates the problem as Aspergillus culture is insensitive). The respective

mortality rates were 65% and 43%. Most are not treated for invasive aspergillosis but

are treated unnecessarily with antibiotics. The scale of the problem is large: In China,

87 per 10,000 people over 40 years of age (an estimated 11,858,000) COPD patients

were admitted to hospital in 2012 (40,41), ~462,000 may have developed invasive

aspergillosis and ~200,000 may have died. Presumably other countries with high

COPD rates such as Hungary, Ireland, and New Zealand face a similar situation.

Asthmatics with frequent exacerbations and/or poorly controlled disease may take

several antibiotic courses a year and some are managed with long-term antibiotics.

Recently, awareness and understanding of ‘fungal asthma’ has increased and a timely

diagnosis and the use of antifungal therapy could substantially reduce antibiotic

prescriptions. Long-term antifungal therapy is efficacious in 60-80% of patients with

fungal asthma, notably allergic bronchopulmonary aspergillosis (ABPA) and severe

asthma with fungal sensitisation (SAFS) (42). Both antibiotic and corticosteroid use

fall with successful antifungal therapy and may reduce hospitalization.

Diagnosis of fungal asthma relies on total and fungal specific IgE testing, or skin

prick testing which is simple to do, yet not often done. Rapid antigen detection with a

lateral flow device might accelerate diagnosis, although no data are published on

utility for sputum, or on COPD patients. Few studies have addressed the role of fungi

in precipitating exacerbations of COPD, although culture, antigen and Aspergillus IgG

antibody all contribute to the diagnosis of chronic and invasive aspergillosis.

Failure to properly diagnose and treat fungal asthma and COPD patients with

Aspergillus colonisation continues to increase the inappropriate use of antibiotics (and

corticosteroids) among these patients. Recognition of fungal infection and/or allergy

with directed antifungal therapy would greatly reduce exacerbations, medical

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consultations and hospital admissions. There is an urgent need to evaluate and

implement both fungal culture and non-culture diagnostics (Aspergillus IgE,

Aspergillus IgG, Aspergillus antigen and PCR) for COPD and asthma exacerbations,

and to diagnose fungal asthma and treat it with antifungals.

Making and excluding the diagnosis of Pneumocystis pneumonia in AIDS

Pneumocystis pneumonia (PCP) in AIDS is often diagnosed empirically based on a

subacute onset of cough, breathlessness out of proportion to the radiological

abnormalities and subtle, bilateral chest X-ray changes, in the context of a low CD4

cell count (Figure 3). Co-trimoxazole (trimethoprim-sulphamethoxazole, Bactrim®,

Septrin®) is the most effective agent for both prevention and therapy of PCP. A low

dose is effective for prophylaxis, but a 3-week course of high and potentially toxic

doses is required for effective therapy. The differential diagnosis of PCP is broader in

children as bacterial pneumonia is more common. If a precise diagnosis could be

achieved in most cases of PCP, much inappropriate use of co-trimoxazole would be

prevented.

Rates of PCP among newly hospitalized adults with advanced HIV infection are

highly variable (<1% to 60%), with rising rates as gross domestic product increases

(43). Without adequate diagnostics available, many patients will unnecessarily

receive high dose co-trimoxazole with or without corticosteroids for 3 weeks. If the

actual number of PCP cases in patients with AIDS is 400,000, then 100,000s may be

given co-trimoxazole unnecessarily, with toxicity rates as high as 90% (5,44). Early

detection and diagnosis of PCP can help prevent unnecessary hospitalization and cost.

Currently bronchoscopy and microscopic examination of bronchoalveloar lavage fluid

is the commonest definitive means of establishing the diagnosis, with a sensitivity of

75-90% depending on the microscopy technique (45). Pneumocystis jirovecii is non-

culturable in routine laboratories and so molecular detection with PCR is commonly

used in Europe with a sensitivity of 95-99% (46). Pneumocystis PCR performed on

expectorated sputum is also effective for detecting P. jirovecii (47-49), yet

infrequently used. In breathless children, PCR on nasopharyngeal aspirates is

currently the only realistic means of establishing the diagnosis. In serum, 1,3 beta-D-

glucan is detectable in nearly all cases of PCP (19), and if negative, effectively rules

out infection. Assuming that 25% of PCP cases are mild, immediate diagnosis and use

of oral therapy will potentially avoid 100,000 admissions to hospital annually, or even

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more if the diagnosis is ruled out and patients are not admitted for unnecessary PCP

therapy (5). Mild PCP responds well to treatment, and prevents progression to

moderate or severe infection.

Provision of rapid Pneumocystis diagnostics will enable early diagnosis, allow

discontinuation of broad spectrum antibiotics if positive and discontinuation of high

dose cotrimoxazole and corticosteroids if negative. Missed PCP diagnoses, obscured

by concurrent bacterial infection, will be minimized. Pneumocystis PCR should be

widely available for all respiratory samples serving large immunocompromised

populations and 1,3 beta-D-glucan in high volume laboratories. These diagnostics will

also definitively improve outcome for non-AIDS immunocompromised patients for

the same reasons as in AIDS.

Other clinical scenarios

We have not addressed multiple other clinical situations in which precise fungal

diagnosis could reduce inappropriate antibacterial prescribing, including cryptococcal

meningitis (Figure 4), overtreatment of Candida found in the respiratory tract and

urine which can be reduced with the use of better markers of infection, under-

diagnosis of invasive aspergillosis in critical care, over-treatment with antifungals of

febrile neutropenia in leukaemia, Aspergillus bronchitis in bronchiectasis, allergic,

chronic and invasive fungal sinusitis and PCP in HIV-negative patients. In all these

clinical settings, inappropriate antibacterial or antifungal prescribing is common,

through lack of adequate fungal diagnostic testing. We have also not addressed either

rapid detection of antifungal resistant fungi (ie Aspergillus terreus, Candida krusei) or

the use of diagnostics to prevent super-infection with high resistance potential fungi,

such as Candida glabrata or Rhizopus oryzae. Antifungal resistance is problematic in

some settings, demands informed prescribing, therapeutic drug monitoring and

development of new antifungal agents (50).

Conclusions

Lack of availability and underuse of non-culture fungal diagnostics results in both

over-prescribing and prescription of unduly long courses of antibacterial agents,

excess empirical use of antifungal agents, and leaves many millions of patients with

undiagnosed fungal infections. It squanders resources. The large scale of the problem,

even in many of the world’s most advanced medical centers, compromises AMR

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control. For many countries, Ministries of Health and private providers of healthcare

should be actively promoting fungal diagnosis to minimize deaths and morbidity from

fungal disease and such efforts will likely have a positive benefit on inappropriate

antibacterial usage and support stewardship programs. Public health authorities need

to embrace both acute and chronic fungal disease areas of considerable need and seize

the opportunity to improve health and preserve what remains of our antimicrobial

toolbox.

Funding: none.

Possible conflicts of interest.

Dr Denning holds Founder shares in F2G Ltd a University of Manchester spin-out antifungal discovery company, in Novocyt which markets the Myconostica real-time molecular assays and has current grant support from the National Institute of Allergy and Infectious Diseases, National Institute of Health Research, NorthWest Lung Centre Charity, Medical Research Council, Astellas and the Fungal Infection Trust. He has acted as a consultant to T2 Biosystems, Astellas, Sigma Tau, Basilea and Pulmocide. In the last 3 years, he has been paid for talks on behalf of Astellas, Dynamiker, Gilead, Merck and Pfizer. He is also a member of the Infectious Disease Society of America Aspergillosis Guidelines and European Society for Clinical Microbiology and Infectious Diseases Aspergillosis Guidelines groups.

Dr. Perlin serves on scientific advisory boards for Merck, Astellas, Scynexis, Matinas and Cidara, and he receives grant support from those companies, as well as from the National Institutes of Health. He is a member of the Clinical & Laboratories Standards Institute (CLSI) Antifungal Working Group. He has been a paid consultant by the Gates foundation. He is an inventor on several patents concerning detection of fungal infections and antifungal drug resistance.

Dr Muldoon has delivered talks on behalf of Pfizer, Gilead, MSD and Astellas, has received an educational grant from Pfizer .

Dr Colombo has received research grants from Pfizer and Astellas, medical education grants from Pfizer, MSD, United Medical and Astellas. He has also been a consultant for MSD, United Medical, and Gilead.

Dr. Chakrabarti has grant support from Department of Biotechnology, Indian Council Medical Research, Government of India, from Pfizer through Asian Fungal Working Group and from MSD through Society of Indian Human and Animal Mycologist and member of European Society for Clinical Microbiology and Infectious Diseases Aspergillosis, Mucormycosis Guidelines groups

Dr Richardson is a consultant and gives talks for Basilea, Astellas, MSD, Pfizer, Gilead Sciences and Associates of Cape Cod.

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Dr Sorrell has received investigator driven grants from Pfizer, Merck, Gilead and have been on their advisory boards in the past.

Author Bio

David Denning is an infectious diseases clinician with expertise in fungal diseases. He is heavily involved in postgraduate teaching and lectures worldwide. He leads LIFE (Leading International Fungal Education) (www.LIFE-Worldwide.org) and is President of the Global Action Fund for Fungal Infections (www.GAFFI.org).

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Figure legends

Figure 1:

Chest radiograph showing bilateral upper lobe chronic pulmonary aspergillosis, easily

mistaken for pulmonary tuberculosis. The blue arrows show the areas of abnormality

in both apices – some pleural thickening and opacification, which is similar although

slightly more obvious to the findings in pulmonary tuberculosis. The orange arrow

shows the trachea pulled to one side by the contraction and fibrosis on that side.

Image from David Denning, all rights reserved.

Figure 2:

CT scan of the thorax showing chronic pulmonary histoplasmosis with bilateral

cavitary infiltrates resembling pulmonary tuberculosis, coccidioidomycosis,

paracoccidioidomycosis and aspergillosis. The abnormal areas are shown by arrows.

Image from Arnaldo Colombo, all rights reserved.

Figure 3:

Chest radiograph showing early subtle abnormalities in both lower lung fields of PCP

in a new diagnosis of AIDS unsuspected in a married man aged 63 years. The two

boxes of magnification illustrate normal lung lung (upper) and infiltrates adjacent to

and behind the heart and overlain by rib (lower). Similar differences between the

upper and lower lobes are seen in the other lung. Image from David Denning, all

rights reserved.

Figure 4:

An ulcerative skin lesion misdiagnosed for weeks as a bacterial infection, which

showed Cryptococccus neoformans on biopsy. Image from Arnaldo Colombo, all

rights reserved.

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