Dear Editor

In a previous issue of this journal, we presented problems of using Bouin’s fixative for immunohistochemical and nuclear morphometric assays on prostatic biopsies(1). We agree with the important comment offered by Berney that Bouin’s fixative could increase the sensitivity for detection of high grade prostatic intraepithelial neoplasia (HGPIN) and prostate cancer. Furthermore, we agree that increased sensitivity could occur at the cost of decreased specificity, as is the case for most diagnostic assays.

Bouin’s fixation was used as a routine fixative for prostatic biopsies at our institution owing to the better nuclear detail offered by this fixative, making the diagnosis of HGPIN easier. Diagnosis of HGPIN assumed greater importance in the ‘sextant biopsy era’, as men with HGPIN had a higher chance of being diagnosed with cancer on repeat biopsies (2). The relevance of finding HGPIN has now decreased owing to extended prostatic biopsy sampling (3). At our institution, there has been switch to formalin for fixing prostatic biopsies for the above two reasons — i.e., decreased predictive value of HGPIN on biopsies, and difficulties with various molecular assays following fixation in Bouin’s fluid. We have noticed a decrease in the prevalence of HGPIN on biopsies following this switch, though a formal analysis has not been performed.

The radical prostatectomy specimens at our institution continue to be fixed with formalin, as they have always been. We agree that using a discrepant fixative such as Bouin’s on biopsies could lead to different patterns of agreement between biopsy and surgical specimens. It indeed would be interesting to analyze the correlation between formalin and Bouin’s fixed specimens to gain a better understanding of how these fixative differences affect the likelihood of diagnosing both HGPIN and cancer.

References

1. Ananthanarayanan V, Pins MR, Meyer RE, Gann PH. Immunohistochemical assays in prostatic biopsies processed in Bouin's fixative. J Clin Pathol 2005;58(3):322-4.

2. Keetch DW, Humphrey P, Stahl D, Smith DS, Catalona WJ. Morphometric analysis and clinical followup of isolated prostatic intraepithelial neoplasia in needle biopsy of the prostate. J Urol 1995;154(2 Pt 1):347-51.

3. Moore CK, Karikehalli S, Nazeer T, Fisher HA, Kaufman RP, Jr., Mian BM. Prognostic significance of high grade prostatic intraepithelial neoplasia and atypical small acinar proliferation in the contemporary era. J Urol 2005;173(1):70-2.

Dear Editor,

Kaushik et al.[1] have demonstrated significantly different gene expression in a small sample of patients with chronic fatigue syndrome. They suggest these observations are consistent with a complex pathogenesis involving T cell activation and abnormalities of neuronal and mitochondrial function, possibly as a result of virus infection or organophosphate exposures.

We suggest that chronic ricketssial infections would be an equally plausible hypothesis, consistent with Kaushik's observations, and worthy of further study. Like viruses rickettsiae are obligate intracellular parasites. Jadin[2] observed in South Africa that, for 500 patients diagnosed with CFS, ME or depression, 80% tested positive to abnormally high levels of ricketssial antibodies using a multiple microagglutination test for antibodies to R Prowazeki, R Mooseri, R Conori, and Coxiella Burnetti ( itself a neo rickettsia).[3]

In the clinical practice of one of the authors (Kemp) all chronically unwell patients, in whom fatigue and/or fibromyalgia are significant, are routinely tested for rickettsia using IgG serology. Over a recent three year period 67% of 750 such patients returned positive serology to at least one of the four rickettsial groups tested, with a titration level of 1/128 or greater considered positive. This data will be submitted for publication at a later time.

Of the seropositive patients only a very small percentage test positive to rickettsiae via real time PCR testing. Moreover very few can recall any incidents in their medical history which would be consistent with an acute rickettsial infection. Most respond significantly to extended antibiotic treatment, mainly using rotating tetracyclines, as described by Jadin (op cit).

While rickettsiae do produce some energy from independent metabolism[4], and can themselves produce small amounts of protein, the energy required for cell division must come from the host cell's mitachondria and the protein sequences from the host cytoplasm. Krausnik's observations of significant perturbation of mitochondrial gene expression might therefore be consistent with chronic rickettsial parasitic infections.

Unlike most viruses ricketsiae can live for quite extended periods in extracellular fluid. Isolated (i.e. non parasitising) rickettsiae can accumulate lysine and lyse erythrocytes, demonstrating their capacity to operate an electron transport system.[5] Their survival in extracellular fluid might contribute to chronic infection and be consistent with Chronic Fatigue patient histories.

Writing in Nature in 1998 Anderson et al.[6] have demonstrated through phylogenetic analyses that R. prowazekii is genetically very closely related to mitochondria. At the time of writing they observed that no other bacteria was as closely related to mitachondria.

While undoubtedly speculative, it is tempting to think that a parasitic bacteria whose replication is absolutely dependent upon the host cell's mitachondria and whose reproductive DNA formation requirements closely match those of the host's mitachondria will be engaged in competition with both the host cell's own energy and own reproductive needs. If this competition resulted in lower levels of mitochondrial capability then a rickettsial etiology for CFS might also explain the functional fatigue seen as the very essence of chronic fatigue syndrome.

G C M Kemp MB BS(Melb) FRACGP, FACNEM
Burke Road Medical Centre
681 Burke Road
Camberwell 3134 Australia

R F Townsend PhD (UNSW) BE (Melb) BBus(Mitchell)
Honorary Research Fellow, Department of Biomolecular Engineering
University of Melbourne,
Parkville 3004 Australia

References

1. N Kaushik, D Fear, S C M Richards, C R McDermott, E F Nuwaysir, P Kellam, T JHarrison, R J Wilkinson, D A J Tyrrell, S T Holgate and J R Kerr Gene expression in peripheral blood mononuclearcells from patients with chronic fatigue syndrome J. Clin. Pathol. 2005;58;826-832

2. C L Jadin, Common Clinical Windows on CFS and Rickettsial Diseases. Journal of Chronic Fatigue Syndrome, 6 no 3 133-145 2000

3. Fiset, P., R. A. Ormsbee, R. Silberman, M. Peacock, and S. H. Spielman.1969. A microagglutination technique for detection and measurement ofrickettsial antibodies. Acta Virol. 13:60–66 and also Kazar, J., R. Brezina, S. Schramek, A. Palanova, and B. Tvrda. 1981 Suitability of the microagglutination test for detection of post-infection and post- vaccination Q fever antibodies in human sera. Acta Virol. 25:235–240.

4. Rickettsial surival in synthetic in vitro solutions have demonstrated that Rickettsial respiration is possible from a wide range of amino acids and sugars,

5. Smith, D. K., and H. H. Winkler. 1977. Characterization of a lysine-specific active transport system in Rickettsia prowazekii.J. Bacteriol. 129:1349-1355.

6. The genome sequence of Rickettsia prowazekii and the origin of mitochondria Siv G. E. Andersson, Alireza Zomorodipour, Jan O. Andersson, Thomas Sicheritz-Ponte ,U. Cecilia M. Alsmark, Raf M. Podowski, A. Kristina, Ann-Soe Eriksson, Herbert H. Winkler Nature vol 396 no 12 November 1998.

Dear Editor,

Kerr has succinctly outlined the events in the past six decades that assisted the clinicians in a judicious care of infectious diseases [1]. During the past decade several automated devices have been marketed that would reduce the turnaround period for bacteriological culture and susceptibility assays. While the majority of investigators found such systems to be so commendable, the recent controlled investigation on clinical outcome of mortality, morbidity and cost on utility of the Vitek 2 system hospitalized patients in the Netherlands is intriguing. In the 1,100-bed tertiary care teaching hospital there was little utility of the Vitek 2 system as against the conventional overnight methods [2].

In all probability, round the clock functioning of microbiology laboratory and frequent dialogues with health care professionals looking after the hospitalized cases would enhance the clinical utility of any conventional or automated microbiology reporting system. That has been apparent during extended clinical microbiology contact in a tertiary care hospital in the Indian capital metropolis of New Delhi.

Sant Parmanand Hospital, a private sector tertiary care 140-bed hospital in the Indian capital metropolis of New Delhi is managed by a benevolent organization that aims to impart first-rate medical care at rational and reasonably priced expenditure. The microbiology laboratory personnel are available round the clock. Rather than vouching for apparently quicker but fairly expensive assays like the Vitek 2 system, a reduction with the turnaround time for microbiology reporting has been attempted. Instead of the conventional overnight gap between inoculation of culture media and recording of growth characters, the hospital would follow a 6-8 hour gap for reading the inoculated culture media. Furthermore, there is a constant dialogue with clinicians regarding any growth of pathogenic bacteria. The antibiotics sensitivity profiles are also communicated verbally to nurses, residents, or the consultants charged with care of patients in the wards. The round-the-clock activities reduced the turnaround time by at least one day. There has been no fiscal cost on the organization or the patients.

The utility of the exercise towards appropriate chemotherapy was evident in two patients with methicillin resistant Staphylococcus aureus (MRSA). A shift from cephalosporins recipe to vancomycin was effective in recovery of two MRSA patients in the surgical intensive care unit [3]. Concurrently, a point-of-care rapid diagnosis on scraping from outpatients with corneal ulcerations has been arranged. Scraped tissues are stained with Leishman and an interim cytological diagnosis towards any fungal, viral or bacterial involvement communicated and discussed with the ophthalmologists [4]. While no systematic evaluation has been carried out on the clinical impact, the clinicians have appreciated the reduced turnaround time. The clinicians have been rather incredulous about the utility of the automation in the microbiology. While automation could be of significant benefit within the laboratory itself, communication with those directly responsible for patient care is vital to improve the quality of healthcare [5]. Last but not least, testing of antimicrobials has been probably the single most important area for close communication between the infectious disease clinician and the clinical microbiologist. Certainly working together of clinical microbiologist and clinicians to resolve basic issues about methods to use for antimicrobial susceptibility testing, agents to test, and the format to use for reporting results would be both cost-efficient and useful towards clinical management of hospitalized and outpatients in tertiary care hospitals. Irrespective of the availability of automated susceptibility assay formats, disk diffusion technology [1] would assist clinicians in offering judicious therapeutic recipe to patients.

ARYA, Subhash C.
AGARWAL, Nirmala
Sant Parmanand Hospital
18 Alipore Road
Delhi- 110054, India
Email: subhashji{at}hotmail.com

References

1. Kerr JR. Antibiotic treatment and susceptibility testing. Journal of Clinical Pathology 2005; 58:786-787

2. Bruins M, Oord H, Bloembergen P, et al. Lack of effect of shorter turnaround time of microbiological procedures on clinical outcomes: a randomised controlled trial among hospitalized patients in the Netherlands. Eur J Clin Microbiol Infect Dis 2005; 24:305-311

3. Arya SC, Kapoor S, Agarwal N, Bhasin R. Re: Evaluation of a disk diffusion method with cefoxitin (30 mG) for detection of Methicillin- resistant Staphylococcus aureus. European Journal of Clinical Microbiology and Infectious Diseases 2004; 23(11): 867-868

4. Anand R, Agarwal N, Arya SC, Shah HK Taneja M. Leishman staining in field diagnosis of corneal ulcer. Saudi Medical Journal 2004; 25:2026-2027

5. Dupont PF. Automation in microbiology: a physician's viewpoint. Am J Med Technol. 1983; 49(5): 323-325

Dear Editor,

We have had experience with the Hemocue and haemoglobin colour chart in India and would like to commnet on the report.

For Hemocue they give a cost per test as $0.75 when in fact the cost of a single cuvette is $0.94 (UK). Further the cost of the instrument in their report is surprisingly low when compared with colourimeters. For example the Jenway 6051 costs £799 and the Hemocue £400. In table 5, equipment costs give a four fold difference when in fact it is only double. Although perhaps of less importance the colour scale is given as zero when each costs $5 (without delivery charge) and, in our own experience, infrequent use needs replacing every 2 years.

In addition there will be the cost of the strips used with the card. We would suggest that adopting the very expensive Hemocue methodfor widespread estimation of haemoglobin will delay the introduction of more useful multi-parameter tests such as semi-automated FBC. We have used the haemoglobin colour scale in various situations in India including rural and slum antenatal clinics, medical camps in remote locations, family planning clinics, etc. In our comparison (unpublished) of the colour scale results against a reference method our findings are almost identical to those given in Table 2.

Dr RP Britt
Former Haematologist at Hillingdon Hospital and visiting Haematologist to Ludhiana.

Address: Woodgate, Latchmoor Grove, Gerrards Cross, Bucks, SL9 8LN.