170 Letters to the Editor

G. ZarfelInstitute of Hygiene, Microbiology and Environmental

Medicine, Medical University of Graz, A-8010 Graz, Austria

A. StoegerAustrian Agency for Health and Food Safety, Institute ofMedical Microbiology and Hygiene, 1226-Vienna, Austria

G. FeierlR.B. Raggam

E. MarthInstitute of Hygiene, Microbiology and Environmental

Medicine, Medical University of Graz, A-8010 Graz, Austria

Accepted 16 December 2008

Available online 18 January 2009

ª 2008 The British Infection Society. Published by Elsevier Ltd. Allrights reserved.doi:10.1016/j.jinf.2008.12.004

Identification of Mycobacterium avium KatG protein(MAV_2753) as a possible serodiagnostic marker forMAC disease

KEY WORDSEnzyme linkedimmunosorbent assay;Humanimmunodeficiency virus;Mycobacterium avium;Diagnosis;Secretory protein

immunodominant protein of w80e85 kDa in 25

To the Editor

Disseminated Mycobacterium avium complex (MAC) infec-tion is a severe complication of advanced HIV disease.1 Thepoor prognosis of disseminated MAC disease2 makes itstimely diagnosis extremely important to initiate theappropriate therapy. However, the various diagnosticmethods, whether conventional (phenotypic identificationof culture and biochemical testing) or new generation(nucleic acid based approaches), so far developed for thedetection and diagnosis of MAC disease, have their ownlimitations and thus prove unsatisfactory.3e5 Therefore,a rapid and simple test for the identification of MACbacteremia is the need of hour. In this regard, serodiagnosison the basis of immunodominant antigens secreted byactively growing bacilli in culture filtrate could be anattractive strategy being an easy to perform, cost effec-tive, rapid6 and highly sensitive technique capable ofdetecting mycobacterial antigens at a concentration of10�9/ml.7 The present study carried out with a goal toidentify M. avium secretory protein(s) of diagnostic signif-icance demonstrated that the KatG protein in M. avium

culture filtrate could be used as a possible serodiagnosticmarker for disseminated MAC disease in AIDS patients.

Various categories of subjects recruited for the presentstudy included HIV seronegative pulmonary TB (PTB)patients (n Z 100) with sputum/bronchoalveolar lavage(BAL) smear positive for AFB, HIV seropositive patients(n Z 54) having disseminated mycobacterial diseasediagnosed on the basis of radiological, clinical or microbi-ological features with CD4þ T cells <100 cells/ml, HIVseropositive patients with no mycobacterial disease(n Z 20) with CD4þ T cell count >100 cells/ml and BCGvaccinated, HIV seronegative healthy volunteers (n Z 20).

M. avium subsp. avium (NCTC 8551; originally isolatedfrom pig liver) was obtained from MTCC, IMTECH, Chandi-garh, India. The comparative analysis of protein profiles ofshort-term culture filtrate (STCF) proteins of M. aviumsubsp. avium, Mycobacterium tuberculosis H37Rv (Mtb) andMycobacterium bovis BCG upon immunoblotting8 withM. avium mouse antisera (1:100) and immunoblotting ofM. avium STCF with different antimycobacterial mouseantisera (1:100), indicated a protein band in the region ofw80e85 kDa, that seemed to be present specifically in M.avium and absent from the rest of the mycobacterialspecies tested. In order to identify and purify theprotein(s) in this region, M. avium STCF was fractionatedby anion exchange column chromatography using a stepgradient of NaCl. This resulted into the elution of an

0 mMelution gradient which showed specific reactivity withM. avium mouse antisera only (data not shown). The250 mM elution gradient was then subjected to high reso-lution preparative SDS-PAGE and electroelution and thefractions containing a pure protein of w82 kDa showingimmunoreactivity specifically with M. avium antisera werepooled and sent to CSU, Colorado, USA for characteriza-tion. The LCeMSeMS analysis of the purified proteinrevealed it to be a protein of molecular weight 81.6 kDa,having 748 amino acids with pI of 4.90. The BLAST searchanalysis identified it as a catalaseeperoxidase protein(KatG) encoded by MAV_2753 that showed >90% homologywith the KatG protein of M. avium 104 (a clinical isolate ofhuman MAC disease) as well as with other members of MAC.Although, it had 65% homology with KatG of M. tuberculosis(Rv1908c), however, the N-terminal 40 amino acids ofM. avium KatG were found to differ 87.5% from N-terminalsequence of Mtb KatG. Immunoreactivity studies of purifiedKatG protein with M. avium and Mtb infected mice serademonstrated the presence of KatG antibodies only inM. avium antisera (Fig. 1a) and confirmed the specificity ofM. avium KatG protein for M. avium infection.

For determining the significance of purified M. aviumKatG protein in the diagnosis of MAC in clinical set-up, thepatients with MAC bacteremia were selected from HIVþ

population with disseminated mycobacterial disease on thebasis of blood culture by lysis centrifugation method,9 fol-lowed by a battery of biochemical tests. Out of 54 bloodsamples of HIV patients with disseminated mycobacterialinfection, 14 samples were mycobacterial culture positiveand of these 14 samples, 10 were MAC and 4 were Mtbpositive on the basis of biochemical tests. When purifiedM. avium KatG protein was subjected to indirect ELISA withthe serum samples (1:100 dilutions) from various study

0

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Figure 1 Serological reactivity of M. avium KatG protein (a) Recognition of M. avium KatG in M. avium infection in mouse model(b) Antibodies against KatG protein in serum samples of HIV seropositive mycobacterial blood culture positive patients (n Z 14).

Letters to the Editor 171

subjects, out of 14 blood culture positive samples, 9samples positive for MAC also gave immunoreactivity withthe KatG protein (Fig. 1b). Four samples that were bloodculture positive for Mtb did not recognize KatG, confirmingthat this protein is specific to M. avium infection. This datademonstrates that the M. avium KatG protein is able todiagnose disseminated MAC disease in HIV infected pop-ulation and the results obtained with antibody-based rapidELISA test correlate with conventional time-consumingblood culture and biochemical tests. Further, M. aviumKatG protein based ELISA in the serum samples from 100pulmonary TB patients demonstrated that 96/100 patientsshowed no reactivity (data not shown), thus confirming thespecificity of M. avium KatG based serodiagnostic assay.Also, the absence of anti KatG antibodies in healthy BCGvaccinated individuals further confirmed the recognition ofthis protein only during active disseminated MAC diseaseand showed that it is not affected by prior BCG vaccina-tion/exposure to environmental mycobacteria.

This study clearly indicates that the development ofM. avium KatG based diagnostic ELISA could be of greathelp to clinicians in timely treatment of disseminated MACdisease in HIV patients. Further studies are in progress toexplore the diagnostic potential of N-terminal KatG of M.avium for the differentiation of disseminated MAC and Mtbinfections in HIV seropositive patients.

Conflict of interest

The authors declare no conflict of interests.

Acknowledgements

We thank the members of TB and Chest Diseases Hospital,Patiala who provided clinical assistance for the study. Wealso thank Dr. Karen Dobos, Colorado State University,Colorado, USA for the LC/MS analysis of KatG protein.

References

1. Corti M, Palmero D. Mycobacterium avium complex infection inHIV/AIDS patients. Expert Rev Anti Infect Ther 2008;6:351e63.

2. Collins TM, Lisby G, Moser C, Chicks D. Results of multiple diag-nostic test for Mycobacterium avium subsp. paratuberculosis in

patients with inflammatory bowel disease and in controls. J ClinMicrobiol 2000;38:4373e81.

3. Arnold Jr LJ, Hammond PW, Wiese WA, Nelson NC. Assay formatsinvolving acridinium-ester-labeled DNA probes. Clin Chem 1989;35:1588e94.

4. Lefmann M, Schweickert B, Buchholz P, Gobel UB, Ulrichs T,Seiler P, et al. Evaluation of peptide nucleic acid-fluorescencein situ hybridization for identification of clinically relevantmycobacteria in clinical specimens and tissue sections. J ClinMicrobiol 2006;44:3760e7.

5. Steadham EJ. Reliable urease test for identification of myco-bacteria. J Clin Microbiol 1979;10:134e7.

6. Kitada S, Kobayashi K, Ichiyama S, Takakura S, Sakatani M,Suzuki K, et al. Serodiagnosis of Mycobacterium avium-complexpulmonary disease using an enzyme immunoassay kit. AmJ Respir Crit Care Med 2008;177:793e7.

7. Kadival GV, Samuel AM, Virdi BS, Kale RN, Ganatara RD. Radio-immunoassay of tuberculosis antigen. Indian J Med Res 1982;75:765e70.

8. Towbin H, Stahelin T, Gordon J. Electrophoretic transfer ofproteins from polyacrylamide gels to nitrocellulose sheets:procedures and some applications. Proc Natl Acad Sci U S A1979;76:4350e4.

9. Salfinger M, Stool EW, Piot D, Heifets L. Comparison ofthree methods for recovery of Mycobacterium aviumcomplex from blood specimens. J Clin Microbiol 1988;26:1225e6.

Kapil GuptaGopal Krishan Khuller

Department of Biochemistry,Postgraduate Institute of Medical Education and Research,

Chandigarh- 160012, INDIA

Ajay WanchuDepartment of Internal Medicine,

Postgraduate Institute of Medical Education and Research,Chandigarh- 160012, INDIA

Suman LaalDepartment of Pathology, NYU School of Medicine,

New York, USA

Romica LatawaIndu Verma*

Department of Biochemistry,Postgraduate Institute of Medical Education and Research,

Chandigarh- 160012, INDIA

172 Letters to the Editor

*Corresponding author. Tel.: þ91 0172 2755180;fax: þ91 0172 2744401.

E-mail address: induvermabio@gmail.com (I. Verma)

Accepted 7 December 2008

Available online 26 January 2009

ª 2008 The British Infection Society. Published by Elsevier Ltd. Allrights reserved.doi:10.1016/j.jinf.2008.12.002

Missedopportunities to diagnose Plasmodium falciparummalaria: Results of a regional service evaluation

Plasmodium falciparum malaria (PFM) is a potentially fatalinfection, and despite the reduction of malaria risk in someparts of the world and the introduction of new drugs, thenumber of people travelling continues to increase andmalaria reports in the United Kingdom (UK) are notdecreasing1 e indeed, malaria accounts for approximately10 deaths per annum in the UK.2 There is a growing body ofevidence highlighting shortcomings in the care provided byhealth services in the UK, including inadequate use ofchemoprophylaxis and public education,3 and recentguidelines from the British Infection Society have re-asserted that early consideration of malaria as a possiblediagnosis is vital for successful management.4 In addition,there is anecdotal evidence of significant delays in thereferral pathway. A regional retrospective service evalua-tion was undertaken in an attempt to assess this objectively.

Confirmed cases of PFM were identified from records atSheffield Teaching Hospitals during the period January 2000to December 2005. All records relating to the episode wereobtained including those from peripheral district hospitaldepartments, and questionnaires were sent to the patient’sregistered General Practitioner (GP). Records weresystematically audited for details regarding demographics,chemoprophylaxis, referral pathways, diagnosis andmanagement.

PFM was diagnosed in 53 adults during the period eval-uated and reliable information for further analysis wasavailable for 39 of these (73.6%). 22 (56.4%) patients orig-inated from countries endemic with PFM, and 17 (43.6%)had travelled for the purpose of visiting friends or relativesin an endemic region (VFR). 34 (87.2%) cases of PFM wereacquired in Sub-Saharan Africa, with the majority of casesfrom West Africa.

Of the 39 patients, only 15 (38.5%) tookany form of malariachemoprophylaxis during their travel abroad. Of the 15, 5were taking appropriate prophylaxis for their destination,according to recommendations in the British National Formu-lary.5 Overall, only 2 patients (5.1%) took appropriateprophylaxisat a sufficientdose for anadequateperiodof time.

The majority of patients (79%) first presented to NHShealth-care services within 2 weeks of arrival in the UK(mean, 11.3; range, 1e61 days). Points of initial contactvaried and included the patient’s own GP (46.2%), emer-gency departments (38.5%), GP Co-operative (5.1%),

Primary Care HCA (Health-Care Assistant e non-medicallytrained) (5.1%), and Primary Care Practice Nurse (2.6%).

8 (20.5%) patients had presented to health professionalson an occasion when, in retrospect, they were symptomaticfor malaria without a referral to hospital or a diagnostictest being performed (ranging from 1 to 6 such encounters).The majority of patients had a first blood-smear taken formalaria diagnosis within 6 h of their initial presentation toa health professional; in 6 cases (15.4%) it was taken over24 h after initial presentation.

17 (43.6%) patients had complicated malaria, as definedby the BIS UK Malaria treatment guidelines.4 Of these 17patients, 1 (5.9%) died and 5 (29.4%) required a period oftreatment on intensive-care. The average length of hospitalstay for surviving patients was 5.45 days (range, 1e28 days).

This evaluation was able to describe the completepatient journey for a significant proportion of the identifiedcases. An almost complete record was available for all thepatients included, with the exception of NHS Direct data.This information was sought but was not made available tous. The demographic results reflect similar findings to thatof a recent Health Protection Agency study, most noticeablyVFR accounting for the majority of cases, and West Africabeing the most common origin of imported infections.1

With increasing media attention following several high-profile cases,6 more pressure is being placed on health-careservices not to miss a PFM diagnosis. This is a difficult andcontroversial area to evaluate, however, this evaluationhighlighted several failures by health-care services whereimprovements are possible:

� Few patients took prophylaxis and those who did rarelyadhered to the full regime. Prophylaxis prescribed wasoften unsuitable for the destination according tonationally available recommendations.� A significant number of patients had encounters with

health professionals whilst symptomatic for malariawithout the diagnosis being considered.� Delays in diagnosis led to a prolonged hospital stay;

patients with a blood film taken within 24 h of presen-tation stayed an average (mean) of 4.45 days,compared to 9.86 days for patients with a film takenafter 24 h.� In one patient who died as a result of PFM (an elderly

Caucasian female travelling to East Africa), the diag-nosis was not considered on their first contact withhealth-care professionals.

While the travel industry have been asked to highlightmalarial prophylaxis,7 improvements also need to be made topre-travel health advice services to ensure that adequatemalaria preventive precautions are recommended by health-care workers, particularly with non-British communities.7

Improvements must include more effective education of thetravelling public about the importance of bite-prevention,the waning immunity to infection after a period away froma malarial locale, and the potential signs and symptomsalerting to a malaria episode. Also, increased awarenessamongst front-line primary care and hospital staff is vital ifmalaria diagnostic services are to improve in the UK, and thismay also involve developing and promoting an enhanced