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Raised urinary neopterin levels and Chlamydia trachomatis infection
I IO
Letters to the Editor
Marker MAK757 569B AI1837 MD011 V010
Mo45 SG20 THI66 Plate I. Patterns by PFGE of SmaI-digested DNAs of various strains of V. cholerae. MAK757, Eltor, Ogawa; 569B Classical, Inaba; A I 1837, OI39 from Bangladesh; MDO I I, OI39 from Madurai, India; VOIo, OI39 from Vellore, India; MO45, OI39 from Madras, India; SG2o, OI39 from Calcutta, India; THI66, Ot39 from Bangkok, Thailand.
serogroup has pandemic potential akin to that of the O ~ serogroup and represents the beginning of the eighth pandemic of cholera.
Department of Microbiology, Faculty of Medicine, Hisao Kurazono, Kyoto University, Kyoto 6o6-oi, Jun Okuda, Japan Yoshifumi Takeda National Institute of Cholera and Enteric Diseases, G. Balakrish Nair Beliaghata, Calcutta, India International Center for Diarrhoeal Disease Research, M. J. Albert, Bangladesh, Dhaka, Bangladesh R. B. Sack Department o/Microbiology and Immunology, Manas Chongsa-nguan, Faculty of Tropical Medicine, Wanpen Chaicumpa Mahidol University, Bangkok, Thailand References i. Ramamurthy T, Garg S, Sharma R et al. Emergence of novel strain of Vibrio cholerae in southern and eastern India. Lancet I993; 341: 703-4. 2. Albert MJ, Siddique AK, Islam MS et al. Large outbreak of clinical cholera due to Vibrio cholerae non-Ox in Bangladesh. Lancet I993; 34I: 704. 3- Chongsa-nguan M, Chaicumpa W, Moolsart P e t al. Vibrio cholerae OI39 Bengal in Bangkok. Lancet I993; 342:430-43 I. 4. Shimada T, Nair GB, Deb BC, Albert MJ, Sack RB, Takeda Y. Outbreak of Fibrio cholerae in India and Bangladesh. Lancet I993; 34I: I346-I347 •
Raised urinary neopterin levels and Chlamydia trachomatis infection Accepted for publication 22 March I994 Sir, We wish to report a link between raised neopterin levels and Chlamydia trachomatis infection in the urine of patients with sterile pyuria.
Letters to the Editor
III
Neopterin (6-D-erythro-trihydroxypropylpterin) is a low molecular weight pyrazino-pyrimidine compound derived biosynthetically from guanosine triphosphate and is a biochemical marker for the activation of cell mediated immunity (CMI). 1'2 It is known that a C M I response is involved in infections caused by C. trachomatis but little is known about the effectiveness of such a response.3 Increased levels of neopterin have been detected in serum and urine from patients with a wide range of microbial infections, autoimmune diseases and certain types of malignancy. 4 T h e most frequently used m e t h o d of quantifying neopterin is by high performance liquid chromatography ( H P L C ) on urine samples which need no pre-treatment and can be stored at - 2 o °C prior to testing. 5 W e have previously examined urine from patients with sterile pyuria for the presence of Chlamydia trachomatis by culture, immunofuorescence ( D I F ) and enzyme immunoassay (EIA) and found a prevalence of io-9% fl and in a recent retrospective study on stored C. trachomatis positive and negative urine samples we have sought a link between raised neopterin levels and C. traehomatis infection, Urine from male and female patients between the ages of 15 and 40 years with more than 2o leucocytes/#l and no apparent infection was routinely examined for C. trachomatis on centrifuged deposits by direct immunofluorescence ( D A K O I M A G E N ) (6) over a six m o n t h period. T w o ml of supernatant was decanted from each centrifuged urine and stored at -- 20 °C for later batch testing for the presence of neopterin. O f the stored aliquots, 40 positive and 180 negative for C. trachomatis were selected and blindly tested for neopterin by reversed phase H P L C using fluorescence detection 353 n m excitation and 438 n m emission wavelengths. Levels of neopterin were calculated using calibration curves set up at the beginning of each run. T h e mean level of neopterin in samples yielding C. trachomatis was 3"55o6#mol/1 with a standard deviation (SD) of I'59 #mol/1 and in the negative samples was I'2176 #mol/1 with a SD of o.8I #mol/1. Statistically neopterin levels were found to be significantly raised in C. trachomatis positive urines in comparison to negative urines (P < o.oooi). T h e levels of neopterin were not influenced by the age range of patients in either the positive or negative samples. On average there was a 3-fold increase in detectable neopterin in the C. trachomatis positive urines compared to the negatives. A n u m b e r of C. trachomatis positive urine samples 4/4o (IO %) had detectable neopterin levels that were below the negative SD for positive samples (I.96o6#mol/1) and 2 6 / I 8 o (I4"4 %) of the negative urine samples had detectable neopterin levels which exceeded the upper SD for negative urines (z'o336/zmol/1). T h e s e discrepancies may be explained by underlying bacterial or viral infections where neopterin levels can increase prior to clinical manifestation 7 or by false negative I M A G E N D I F which is not as sensitive as EIA 6 but in this retrospective blind study we were unable to investigate further. Obviously, elevated neopterin levels in biological fluids are not specific for chlamydial infections, however, levels observed in this study and the correlation between the presence of C. trachomatis and raised neopterin levels, indicates that neopterin has the potential to be used as an additional biochemical marker for the presence of C. trachomatis in urine and should be investigated further.
Department of Microbiology, Dudley Road Hospital, Birmingham BI8 7QH, U.K.
L. Kelly ft. M. Woodcock R. S. Matthews R. Wise References
i. Kaufman S, Fisher DB. Acid hydroxylases. In: Molecular Mechanism of Oxygen Activation. New York, Academic Press, I974: 285-369. 2. Huber C, Batchelor JR, Fuchs D, et al. Immune response-associated production of
II2
3. 4. 5. 6. 7-
Letters to the Editor neopterin. Release from macrophages primarily under control of interferon-gamma. J Exp Med I984; I6o: 3IO-316. Morrison RP. Immune responses to Chlamydia are protective and pathogenic. Proceedings of the 7th International Symposium on Human Chlamydia Infections (British Columbia) June I99o: Cambridge University Press, I63-I72. Wachter H, Fuchs D, Hausen A, Reibnegger G, Werner ER: Neopterin as marker for activation of cellular immunity: Immunological basis and clinical application. Adv Clin Chem I989; 27: 8I-I4I. Hausen A, Fuchs D, Konig K, Wachter H. Determination of neopterin in human urine by reversed-phase high performance liquid chromatography. J Chromatog I982; z27: 6I-7O. Matthews RS, Bonigal SD, Wise R: Sterile pyuria and Chlamydia trachomatis. Lancet I99O; 336: 385. Hansen A, Fuchs D, Reibnegger G, Werner ER, Wachter H. Neopterin in clinical use. Pteridines I989; I: 3-IO. Human infection with
Cyclospora
Accepted for publication z2 March I994 Sir, T h e r e have been a n u m b e r of reports of diarrhoea caused by a newly-described organism, known initially as a cyanobacterium or coccidia-like body (CLB), and first described by Ashford. 1 It has recently been proposed that the organism be named Cyclospora cayetanensis. 2 Cyclospora has been implicated as a cause of protracted diarrhoea in individuals who live in, or have visited, the United States, the Caribbean Islands, Central and South America, Southeast Asia, and Eastern Europe. Outbreaks have been reported in NepaP and Chicago. 4 We have screened I333 faecal samples for the presence of Cyclospora oocysts using smears stained by the modified Ziehl-Neelsen (ZN) method. 5 T h e organism has also been shown to autofluoresce under ultra-violet light 6 although this characteristic was not used for screening purposes. Cyclospora oocysts were detected in the faeces of one female and one male patient. Routine bacteriological investigations were negative in both patients. T h e oocysts were identified by their size (about 8-9 # m in diameter), their variable staining with the modified Z N stain and their internal structure. T h e oocysts from both patients fluoresced weakly under ultra-violet light. T h e first patient was a 29-year-old air hostess who presented with a Io-day history of diarrhoea, nausea, vomiting, anorexia, weight loss and colicky abdominal pain which started whilst on holiday in Indonesia. H e r illness commenced after she had been in Indonesia for I I days. H e r symptoms started to abate after four weeks of illness. Oocysts were still being excreted in low numbers 38 days after the onset of symptoms. T h e second patient was a 29-year-old male who lived in Indonesia but had also recently travelled to France. Cysts of Blastocystis hominis were also present in his faeces. T h e patient presented with an I I-day history of similar symptoms to the first patient, but without vomiting. Dyspepsia and fever were also reported. T h e patients' wife and baby son experienced similar symptoms although faecal specimens were not received from them. Both patients had been careful to avoid drinking untreated water. We found that the modified Z N stain was an effective m e t h o d for the screening of faecal samples for the presence of Cyclospora. T h e organisms were readily identified by their size, staining characteristics and internal structure. Cyclospora should be considered in individuals with unexplained protracted diarrhoea who have travelled to the above mentioned countries.
Letters to the Editor
Marker MAK757 569B AI1837 MD011 V010
Mo45 SG20 THI66 Plate I. Patterns by PFGE of SmaI-digested DNAs of various strains of V. cholerae. MAK757, Eltor, Ogawa; 569B Classical, Inaba; A I 1837, OI39 from Bangladesh; MDO I I, OI39 from Madurai, India; VOIo, OI39 from Vellore, India; MO45, OI39 from Madras, India; SG2o, OI39 from Calcutta, India; THI66, Ot39 from Bangkok, Thailand.
serogroup has pandemic potential akin to that of the O ~ serogroup and represents the beginning of the eighth pandemic of cholera.
Department of Microbiology, Faculty of Medicine, Hisao Kurazono, Kyoto University, Kyoto 6o6-oi, Jun Okuda, Japan Yoshifumi Takeda National Institute of Cholera and Enteric Diseases, G. Balakrish Nair Beliaghata, Calcutta, India International Center for Diarrhoeal Disease Research, M. J. Albert, Bangladesh, Dhaka, Bangladesh R. B. Sack Department o/Microbiology and Immunology, Manas Chongsa-nguan, Faculty of Tropical Medicine, Wanpen Chaicumpa Mahidol University, Bangkok, Thailand References i. Ramamurthy T, Garg S, Sharma R et al. Emergence of novel strain of Vibrio cholerae in southern and eastern India. Lancet I993; 341: 703-4. 2. Albert MJ, Siddique AK, Islam MS et al. Large outbreak of clinical cholera due to Vibrio cholerae non-Ox in Bangladesh. Lancet I993; 34I: 704. 3- Chongsa-nguan M, Chaicumpa W, Moolsart P e t al. Vibrio cholerae OI39 Bengal in Bangkok. Lancet I993; 342:430-43 I. 4. Shimada T, Nair GB, Deb BC, Albert MJ, Sack RB, Takeda Y. Outbreak of Fibrio cholerae in India and Bangladesh. Lancet I993; 34I: I346-I347 •
Raised urinary neopterin levels and Chlamydia trachomatis infection Accepted for publication 22 March I994 Sir, We wish to report a link between raised neopterin levels and Chlamydia trachomatis infection in the urine of patients with sterile pyuria.
Letters to the Editor
III
Neopterin (6-D-erythro-trihydroxypropylpterin) is a low molecular weight pyrazino-pyrimidine compound derived biosynthetically from guanosine triphosphate and is a biochemical marker for the activation of cell mediated immunity (CMI). 1'2 It is known that a C M I response is involved in infections caused by C. trachomatis but little is known about the effectiveness of such a response.3 Increased levels of neopterin have been detected in serum and urine from patients with a wide range of microbial infections, autoimmune diseases and certain types of malignancy. 4 T h e most frequently used m e t h o d of quantifying neopterin is by high performance liquid chromatography ( H P L C ) on urine samples which need no pre-treatment and can be stored at - 2 o °C prior to testing. 5 W e have previously examined urine from patients with sterile pyuria for the presence of Chlamydia trachomatis by culture, immunofuorescence ( D I F ) and enzyme immunoassay (EIA) and found a prevalence of io-9% fl and in a recent retrospective study on stored C. trachomatis positive and negative urine samples we have sought a link between raised neopterin levels and C. traehomatis infection, Urine from male and female patients between the ages of 15 and 40 years with more than 2o leucocytes/#l and no apparent infection was routinely examined for C. trachomatis on centrifuged deposits by direct immunofluorescence ( D A K O I M A G E N ) (6) over a six m o n t h period. T w o ml of supernatant was decanted from each centrifuged urine and stored at -- 20 °C for later batch testing for the presence of neopterin. O f the stored aliquots, 40 positive and 180 negative for C. trachomatis were selected and blindly tested for neopterin by reversed phase H P L C using fluorescence detection 353 n m excitation and 438 n m emission wavelengths. Levels of neopterin were calculated using calibration curves set up at the beginning of each run. T h e mean level of neopterin in samples yielding C. trachomatis was 3"55o6#mol/1 with a standard deviation (SD) of I'59 #mol/1 and in the negative samples was I'2176 #mol/1 with a SD of o.8I #mol/1. Statistically neopterin levels were found to be significantly raised in C. trachomatis positive urines in comparison to negative urines (P < o.oooi). T h e levels of neopterin were not influenced by the age range of patients in either the positive or negative samples. On average there was a 3-fold increase in detectable neopterin in the C. trachomatis positive urines compared to the negatives. A n u m b e r of C. trachomatis positive urine samples 4/4o (IO %) had detectable neopterin levels that were below the negative SD for positive samples (I.96o6#mol/1) and 2 6 / I 8 o (I4"4 %) of the negative urine samples had detectable neopterin levels which exceeded the upper SD for negative urines (z'o336/zmol/1). T h e s e discrepancies may be explained by underlying bacterial or viral infections where neopterin levels can increase prior to clinical manifestation 7 or by false negative I M A G E N D I F which is not as sensitive as EIA 6 but in this retrospective blind study we were unable to investigate further. Obviously, elevated neopterin levels in biological fluids are not specific for chlamydial infections, however, levels observed in this study and the correlation between the presence of C. trachomatis and raised neopterin levels, indicates that neopterin has the potential to be used as an additional biochemical marker for the presence of C. trachomatis in urine and should be investigated further.
Department of Microbiology, Dudley Road Hospital, Birmingham BI8 7QH, U.K.
L. Kelly ft. M. Woodcock R. S. Matthews R. Wise References
i. Kaufman S, Fisher DB. Acid hydroxylases. In: Molecular Mechanism of Oxygen Activation. New York, Academic Press, I974: 285-369. 2. Huber C, Batchelor JR, Fuchs D, et al. Immune response-associated production of
II2
3. 4. 5. 6. 7-
Letters to the Editor neopterin. Release from macrophages primarily under control of interferon-gamma. J Exp Med I984; I6o: 3IO-316. Morrison RP. Immune responses to Chlamydia are protective and pathogenic. Proceedings of the 7th International Symposium on Human Chlamydia Infections (British Columbia) June I99o: Cambridge University Press, I63-I72. Wachter H, Fuchs D, Hausen A, Reibnegger G, Werner ER: Neopterin as marker for activation of cellular immunity: Immunological basis and clinical application. Adv Clin Chem I989; 27: 8I-I4I. Hausen A, Fuchs D, Konig K, Wachter H. Determination of neopterin in human urine by reversed-phase high performance liquid chromatography. J Chromatog I982; z27: 6I-7O. Matthews RS, Bonigal SD, Wise R: Sterile pyuria and Chlamydia trachomatis. Lancet I99O; 336: 385. Hansen A, Fuchs D, Reibnegger G, Werner ER, Wachter H. Neopterin in clinical use. Pteridines I989; I: 3-IO. Human infection with
Cyclospora
Accepted for publication z2 March I994 Sir, T h e r e have been a n u m b e r of reports of diarrhoea caused by a newly-described organism, known initially as a cyanobacterium or coccidia-like body (CLB), and first described by Ashford. 1 It has recently been proposed that the organism be named Cyclospora cayetanensis. 2 Cyclospora has been implicated as a cause of protracted diarrhoea in individuals who live in, or have visited, the United States, the Caribbean Islands, Central and South America, Southeast Asia, and Eastern Europe. Outbreaks have been reported in NepaP and Chicago. 4 We have screened I333 faecal samples for the presence of Cyclospora oocysts using smears stained by the modified Ziehl-Neelsen (ZN) method. 5 T h e organism has also been shown to autofluoresce under ultra-violet light 6 although this characteristic was not used for screening purposes. Cyclospora oocysts were detected in the faeces of one female and one male patient. Routine bacteriological investigations were negative in both patients. T h e oocysts were identified by their size (about 8-9 # m in diameter), their variable staining with the modified Z N stain and their internal structure. T h e oocysts from both patients fluoresced weakly under ultra-violet light. T h e first patient was a 29-year-old air hostess who presented with a Io-day history of diarrhoea, nausea, vomiting, anorexia, weight loss and colicky abdominal pain which started whilst on holiday in Indonesia. H e r illness commenced after she had been in Indonesia for I I days. H e r symptoms started to abate after four weeks of illness. Oocysts were still being excreted in low numbers 38 days after the onset of symptoms. T h e second patient was a 29-year-old male who lived in Indonesia but had also recently travelled to France. Cysts of Blastocystis hominis were also present in his faeces. T h e patient presented with an I I-day history of similar symptoms to the first patient, but without vomiting. Dyspepsia and fever were also reported. T h e patients' wife and baby son experienced similar symptoms although faecal specimens were not received from them. Both patients had been careful to avoid drinking untreated water. We found that the modified Z N stain was an effective m e t h o d for the screening of faecal samples for the presence of Cyclospora. T h e organisms were readily identified by their size, staining characteristics and internal structure. Cyclospora should be considered in individuals with unexplained protracted diarrhoea who have travelled to the above mentioned countries.