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Diagnosis, Prevalence and Drug Resistance of Mycobacteria in HIV-Positive and HIV-Negative Patients of an Urban Population in Germany
Zbl. Bakt. 277, 119-128 (1992) © Gustav Fischer Verlag, Stuttgart/New York
Diagnosis, Prevalence and Drug Resistance of Mycobacteria in HIV-Positive and HIV-Negative Patients of an Urban Population in Germany RICHARD RATEI, WALEED ZAKI, JUTTA WAGNER, and HELMUT HAHN Institut fiir Medizinische Mikrobiologie und Infektionsimmunologie, Freie Universitat Berlin, 1000 Berlin, Germany
With 1 Figure· Received August 2, 1991 . Revision received November 22, 1991 . Accepted Janu ary 21, 1992
Summary In a survey of 3004 clinical specimens from 1112 HIV-negative and 679 clinical specimens from 140 HIV-positive patients which were submitted to our mycobacter iallaboratory, we have analysed the different diagno stic approaches concerning mycobacterial disease in these two populations. We have assessed specimen-rype, culture result, microscopical diagnosis and prevalence in relation to HIV status. Isolation rates of mycobacteria were highest in blood culrures from HIV-positive patients . 10 out of 140 HIV-positive patients had positive mycobacterial culture results. All 10 had positive blood cultures, but only three of them were positive for M. tub erculosis. Zusammenfassung Anhand von 3004 mikrobiol ogischen Proben von 1112 HIV-negat iven Parienten und von 679 mikrobiologischen Proben von 140 HIV-positiven Patienten wurden die unterschiedlichen diagnostischen Vorgehensweisen beztiglich einer Mykobakterieninfektion bei diesen beiden Populationen unrersucht. Hierb ei wurden sowohl die Art des eingesandten Materials, das Kulturergebnis, die mikro skopische Diagnose, als auch die Pravalenz berticksichtigt und in Bezug zum HIV-Statu s gesetzt. Die hochste kulturelle Nachweisrate fand sich in Blutkulturen von Hl'V-Paticnren. Von insgesamt 140 HIV-positiven Patienten konnte bei 10 Patienten ein kultureller Nachwei s gefuhrt werden. Bei jedem dieser 10 Patienten konnten Mykobakterien in der Blutkultur nachgewiesen werden. Nur drei dieser Isolate waren M. tub erculosis. Introduction Tuberculosis has remained to be a major public health problem with an estimate of 16 million prevalent cases, one billion infected and about 8 million new cases of
R. Ratei, W.Zaki, J. Wagner, and H. Hahn
120
tub erculosis occurring annually thr oughout the world (11). The magn itude and dynamics of the world's tube rculosis problem are mainly reflected by the situation in the developing countries where the annual risk of tuberculosis infection has hardly decreased at all since 1900. In contrast, there has been a steady decline in the industrialized world down to an annua l risk of tuberculosis infection of below 0.05 % (11). The dramatic redu ction in morbidity and mortality from tub erculosis in the indu strialized countries can be attributed mainly to the socioeconomic development whi ch brought better living conditio ns, pa rticularly with regard to general health care and nutrition and the introdu ction of tu berculosta tic chemotherapy. Nonetheless, eradi cation of tub erculosis or mycobacterial diseases in the indu strialized countries is far from complete (5, 9). The total annual incidence of tub erculosis in the Federal Republ ic of Germany (FRG) declined from 48.2 11 00.000 in ] 978 to 21.9 /100.000 in 1988. In the first seven year s of th is decade, the tot al incidence dropped annually by a mean of 3.38 cases/ 100,000. During the follow ing four years, the total incidence decreased only by an annual mean of 1.96 cases/lOO,OO, leading to a flattening of the form erly steep decline of the total incidence of tuberculosis (Fig. 1). This development was almost paralleled by the incidence rates for first infections. Only the reinfection rates have been constantly decreasing with O.2-0.9/100,OOO/year (12).
60
...... nI
u
-
~
0
Gl
~
Gl
C
'u
40
0
0
~
~
.5 Cii
Total
20
First infection
Gl III
III
u
Reinfection
o 78
79
80
81
82
83
84
85
86
87
88
time (years)
Fig. 1. Incidence of tuberculosis in the Federal Republic of Germany between 1978 and 1988 (12).
Th at gives suppo rt to the notion that the eradication of tuberculosis has come to a poin t where first infections cannot be prevented with the same efficacy like they had been unt il 1984. The rate of reinfection s remained constant dur ing the same period. With the advent of HIV and AIDS, new clinical and epidemiological aspects have to be considered in the estimation of tub erculosis and mycobacterial diseases. Especially the occurrence of " mycobacteria other than tuberculosis" (M OTT ) in clinical specimens from patients with AIDS has to be evaluated in the light of the path ogenesis of mycobacteria-associated symptoms like dia rrhea and wasting (10, 7).
Mycobacteria in HIV-Positive and HIV-Negative Patients
121
In the following report we have summarized the data gathered by our mycobacterial laboratory to present an analysis on the prevalence, the drug resistance and sensitivity and the identification of mycobacterial isolates from a heterogeneity of clinical specimens from two groups of patients thought to be representative of an average size mycobacterial laboratory in the urban area of Berlin.
Material and Methods Clinical specimens. Between July 1", 1991 and December 31", 1991 3683 clinical specimens from Hl V-positive and HIV-negative patients were routinely subjected to mycobacterial diagnosis in our laboratory. All patients were either in- or outpatients from two major and various minor hospitals belonging to the western boroughs of Berlin. HIV status, origin and type of specimen were assesed according to informations given by the requesting clinician. Culture and identification. Depending on the type of specimen and the clinical situation, the materials were concentrated by centrifugation, pretreated and then submitted to microscopy and culture. Microscopy is routinely performed with either Auramin or Ziehl-Neelsen staining, or both. Three different culture media are inoculated separately: LoewensteinJensen, Gottsacker and Stonebrink. Inoculated cultures are incubated for 8-12 weeks at 37°C in a COrenriched atmosphere and checked weekly for growth. For further identification, cultural isolates are routinely evaluated by the following reactions: colony morphology, growth at 25 °C/42°C, catalase activity, niacin production, nitrate reduction, urease Tween 80 hydrolysis, presence of arylsulphatase, pyrazinamidase, pigment production, iron uptake, resistance to INH I and TCH 2 • Testing for drug resistance and sensitivity. Using a combination between the proportion method according to Canetti and the MIC-method resistance and sensitivity to the following tuberculostatic drugs were routinely tested: isoniazid (0.25-1.0 ug/ml), streptomycin (4.0-8.0 ug/ml), ethambutol (1.0-2.0 ug/rnl) , rifampicin (8.0-32.0 ug/ml), capreomycin (16.0-32.0 Itg/ml), cycloserine (16.0-32.0 ug/ml) and prothionamide (16.0-32.0 ug/ml) (4). Data processing and statistics. All data, including patient identification, type of specimen, microscopy, HIV status, culture period, species diagnosis and drug resistance were gatherd in a data bank on an EXL computer (© 1988 Prime Computer) running under the Unix operating system V Release 3.1. Data processing and statistics were performed with P-STAT 8, version 2.11 (© 1989, P-STAT, Inc.) software.
Results Specimens and culture results within patient status We screened 3683 clinical specimens from 1252 patients, 140 (11.2%) of whom were known to be HIV-positive and 1112 (88.8%), HIV-negative. With 3004 (81.6%) specimens from HIV-negative patients the specimen/patient ratio as a measure for the diagnostic surveillance scored 2.7 whereas in the HIV-positive population a mean of 4.9 specimens/patient is indicative of the different and much more vigilant diagnostic surveillance afforded to these patients (Table 1). The heterogeneity of clinical specimens from these two populations and the cultural results have been summarized in Table 2.
I 2
isoniazid (10 Itg ml- I ) thiophene-2-carboxylic acid hydrazide (1 Itg ml")
122
R. Ratei, W. Zaki,
J. Wagner, and H. Hahn
Table 1. HIV status, number of patients and specimens and specimen/patient ratio Status
Patients
Specimens
specimens! patient
HIV-negative HIV-positive
1112 (88.8%) 140 (11.2%)
3004 (81.6%) 679 (18.4%)
2.7 4.9
Table 2. Heterogeneity of clinical specimens sorted according to patients status and cultural result. The original 70 different specimen types have been summarized into 20 categories: B.secret. = Bronchialsecretion, B.lavage = Bronchiallavage, P.fluid = Pleural fluid, Gaspirate = gastric aspirate, CSF = cerebrospinal fluid, Misc. = Miscellaneous, ns. = not specified Culture negative Specimen Sputum B.secret. B.lavage P.fluid G.aspirate Ascites Blood CSF Lymphnode Urine
Total
HIV status positive negative
599 40 168 165 173 17 205 87
507 37 125 160 158 16 19
12
1341
10 1245
39 3 37 3 10 1 163 15 1 83
2 11
1 3
8
72
Culture positive HIV status negative positive 12
1
4 2 4
2 1 23
1 9
4
Biopsies
Liver Duodenum Ileum Colon Sigma Rectum
1
2
1
1
6 7 13
2 5 10
4 2 3
Stool Swabs Misc. ns.
597 97 166 15
363 85 126 15
228 5 34
1 5 6
5 2
Total
3683
2960
640
44
39
Obvious differences are seen in the distribution of specimens obtained for the diagnosis of mycobacterial disease from these two populations. They clearly reflect the underlying pathogenetic principles of tuberculosis in the HIV-negative not immunocompromised patient and mycobacterial disease due to ubiquitous mycobacteria in the HIV-positive immunocompromised patient. Thus the diagnostic mainstay in the
Mycobacteria in HIV-Positive and HIV-Negative Patients
123
HIV-positive population is carried by culturing stool and blood specimens, whereas in the HIV-negative population, specimens from the respiratory tract and urine are much more common. For the HIV-negative patients, specimens most commonly associated with positive cultural results are sputum, urine and swabs from various sites as compared to blood and stool for HIV positive patients. The exact origins of all 83 clinical specimens which have been culturally positive for mycobacteria are shown in Table 3.
Table 3. Depiction of clinical specimens and positive culture results according to HIV status. Swabs ns. = swabs not specified, wound sw. = wound swab, punctate ns. = punctate not specified, tissue ns. = tissue without any further specification, gastric a. = gastric aspirate, wound sec. = wound secretion, cystic f. = cystic fluid, biopsy ns. = biopsy not specified, bone m. = bone marrow Specimen Swab ns. Wound sw. Bronchial!. Blood Tissue ns. Bone m. Lymphnode Hastric a. Biopsy ns.
HIV status negative positive 3 4
2 2 23
1 1 1 4 2
Specimen
HIV status negative positive
Liver biopsy Punctate ns. Pleural fluid Pus Sputum Stool Wound sec. Cystic f. Urine
1 2 2 12 1 1 1 9
1
1 5 4
The overall isolation rate of 83 (2.25%) positrve cultures from 3683 specimens composed of 70 different specimen types refers to only 19 different specimen types. The overall isolation rate of 35 out of 1252 patients (2.8%) includes 25 HIV-negative patients resulting in a prevalence of 2.25% for this population and 10 HIV-positive patients with a significantly (Yates X 2 = 8.38; pr = 0.004) higher prevalence of 7.14% (Table 4).
Table 4. Prevalence of mycobacteria in HIV-positive patients is significantly higher than in HIV negative patients (Yates X 2 = 8.38; pr = 0.0004) Status
Patients
Positive culture
Prevalence
HIV-negative HIV-positive
1112 140
25 10
2.25% 7.14%
Total
1252
35
2.8%
124
R. Ratei, W. Zaki, J. Wagnet, and H. Hahn
Results of microscopy and culture and patient status 3
Of 3683 clinical specimens, 1264 (34.3 % ) were subjected to microscopical diagnosis with only 1.9 % (n = 25) positive results. Microscopy was not done in 65.7% (n = 2419 ) of all specimens due to insufficiency of specimen type or specimen quantity. From 83 culturally positive specimens in both groups of patients, only 54 had been microscoped of which 44 were false-negative, i.e., negative microscopy and positive cultural result . Onl y 10 specimens had positive microscopic results which were confirmed later on by positive culture result. Thus , sensitivity of microscop y is expressed by the ratio of trul y positive microscopies (n = 10) and the number of positive culture results (n = 54 ). .. . truly positive microscopies 10 0 18 .. = - = . Senstuutty = postttue culture results 54 Truly negative microscopies were attested by negative culture results in 777 specimens of HIV-negative patients and in 418 specimens of HIV-= positive patients. In 15 specimens, positive microscop y was not confirmed by the culture result. Accordingly, specificity of microscopy is exp ressed by the ratio of truly negative microscopies (n = 1195 ) and the number of negative culture results (n = 1210 ). S eciticit = truly ne?ative microscopies = 1195 = 0.99 P I' Y negatt ue culture results 1210 The accurac y of microscop y in detecting mycobacteria is expressed by the number of truly positive microscopies divided by the number of all positive microscopies including tho se which were not been confirmed later on by a positive culture result. I
_
A ccurac) -
trul y positiv e microscopies II .. . . a postttue mt croscopies
10 = -25 = 0040
Species identification and pati ent status"
In the group of HIV-negative patients, we cultured M. avium from specimens of four pati ents, M. bovis and M. gordonae in one patient each and M. tuberculosis in 19 patients, which amounted to a total of 25 isolates in this group. Only two species were isolated in the HIV-positive group of wh ich 70% (n = 7) were M. avium and 30 % (n = 3) were M. tuberculosis. Drug resistance and sensitivity:
The resistance of the 11 M. avium strains was in accordance with the well known multiple resistance of atypical mycobacteria to primary antituberculosis drugs like INH 6 , streptomycin, ethambutol and rifampicin. All M. avium strains showed susceptiblity to cycloserine at a concentration of between 16.0 and 32.0 ug/ml. M. tuberculosis strains showed consistent susceptibility to all the tested primary antituber-
see Table 5 see Table 6 5 see Table 7 (, Isoniazid 3
4
Mycobacteria in HlV-Positive and HlV-Negative Patients
125
Table 5. Patient status within culture result. Cell contents are; Total N = cell counts, Row Pet = row percent, Col Pet = column percent, and Tot Pet = percent of total N Culture negative
Culture positive
Microscopy
Total
H1V status negative positive
HIV status negative positive
no Total N Row Pet Col Pct Tot Pct
2419 100.0 65.7 65.7
2176 90.0 73.5 59.1
214 8.8 33.4 5.8
22 0.9 50.0 0.6
7 0.3 17.9 0.2
negative Total N Row Pct Col Pet Tot Pct
1239 100.0 33.6 33.6
777 62.7 26.2 21.1
418 33.7 65.3 11.3
18 1.4 40.9 0.5
26 2.1 66.7 0.7
positive Total Row Pet Col Pet Tot Pct
25 100.0 0.7 0.7
7 28.0 0.2 0.2
8 32.0 1.2 0.2
4 16.0 9.1 0.1
6 24.0 15.4 0.2
Total N Row Pct Col Pet Tot Pet
3683 100.0 100.0 100.0
2960 80.4 100.0 80.4
640 17.4 100.0 17.4
44 1.2 100.0 1.2
39 1.0 100.0 1.0
Table 6. Species identification and patient status of the 35 patient isolates on the basis of 83 culturally positive specimens Patient status H1V negative H1V positive Total
M. avium
4 7 11
M. bovis
M. gordonae
M. tuber.
Total
1
1
19 3
25 10
21
35
culosis drugs as well as to cycloserine and PTH 7 • Resistance to streptomycin at a concentration of 4 to 8 ug/ml was found in two strains. Two other strains only showed sensitivity to streptomycin at a concentration of 8 ug/ml and not at 4 ug/rnl. These two strains were considered to be in the borderline of resistance.
7
Prothionamide
126
R. Ratei, W.Zaki, J.Wagner, and H. Hahn
Table 7. Drug resistance and sensitivity of 35 isolates of mycobacteria. INH = isoniazid, PAS = p-aminosalicylic acid, PTH = prothionamide Antibiotic
M. autum
M. bovis
M. gordonae
M. tuber.
Total
INH
resistant sensitive
11 22
12 23
Streptomycin
borderline resistant sensitive
10 1
1
2 2 18
20
22
11 24
22
11 24
5 22
11 24
22
1 34
22
35
2 13
Ethambutol
resistant sensitive
11
Rifampicin
resistant sensitive
11
1
Capreomycin
resistant sensitive
11
Cycloserine
resistant sensitive
1 11
PTH
sensitive
11
1
Discussion The diagnosis of tuberculosis does not only rely upon the isolation and identification of acid-fast rods in clinical specimens alone but is mainly based on clinical grounds. In fact, tuberculosis of the respiratory system in the Federal Republic of Germany in 1986 and 1987 was diagnosed in 50% of cases (10.71100,000) on clinical grounds alone, without the isolation of tuberculosis bacteria (12). Nonetheless, it has been shown recently that for the eradication of tuberculosis it is of paramount importance to isolate and identify the mycobacterial strain in each case in order to track down and control infection routes (6). With the advent of HIV infections, the occurrence of atypical mycobacteria in clinical specimens must be borne in mind and their pathogenetic role in the immunocompromised host has to be meticulously evaluated (3). As one can see from the specimen-patient ratio (Tabl 1), the surveillance and diagnostic coverage by taking specimens to be examined for mycobacteria is much better
Mycobacteria in Hlv-Positive and HIV-Negative Patients
127
for the HIV-positive population than it is for the HIV-negative population. The assessment of prevalence rates of positive mycobacterial culture results in these two populations (Table 4) must therefore be considered with reservation because of different diagnostic approaches. Thus, the question arises whether the rate of tuberculosis cases with positive isolation of M. tuberculosis in the HIV-negative population would increase with a more stringent diagnostic approach including more and better specimens and enhanced detection methods, e.g. the polymerase chain reaction (8, 1). In the HIV-positive group, the isolation rate of mycobacteria from blood cultures was particularly high: of 140 patients in whom multiple and various cultures were performed, three patients had positive blood cultures for M. tuberculosis and seven, for M. avium. This accounts for an overall isolation rate of mycobacteria in blood cultures of 7.14%, of 0.21 % for M. tuberculosis and of 0.5% for M. avium. This is in contrast to the isolation rates mentioned by Bonza et al. who have described a total of 67 cases of tuberculosis among 100 patients with AIDS and an isolation rate of M. tuberculosis from blood cultures of 64% in these patients (2). In our survey, M. tuberculosis was cultured in only three HIV -positive patients out of 140 in whom blood cultures had been performed. The low isolation rate of M. tuberculosis in HIV-positive patients in this survey as compared to Bonza et al. can most certainly be attributed to a lower incidence of tuberculosis in Germany and to a different composition of the HIVpositive population with regard to socioeconomic status, homosexuality and drug abuse.
References 1. Boeddingbaus, T. Rogall, T. Flohr, H. Bloecker, and E. C. Boettger: Detection and identification of mycobacteria by amplification of rRNA. J. Clin. Microbiol. 28 (1989) 1751-1759 2. Bonza, E., C. Martin-Scapa, j. C. L. Bernaldo de Quiros et al.: High prevalence of tuberculosis in aids patients in spain. Europ. J. Clin. Microbiol. Infect. Dis. 7 (1988) 785-788 3. Braun, M. M., R. H. Byers, W. L. Heyward et al.: Acquired immunodeficiency syndrom and extrapulmonary tuberculosis in the united states. Arch. Intern. Med. 150 (1990) 1913-1916 4. Canetti, G.: Mycobacteria: Laboratory methods for testing drug sensitivity and resistance. Bull. Wid Hlth Org. 29 (1963) 5565-578 5. Gracey, D. R.: Tuberculosis in the world today. Mayo Clin. Proc. 63 (1988) 1251-1255 6. Gross, T. P., P. R. Silverman, A. B. Bloch et al.: An outbreak of tuberculosis in rural Delaware. Amer. J. Epidem. 129 (1989) 362-371 7. Guarner, j., C. del Rio, and B. Slade: Tuberculosis as a manifestation of the acquired immunodefiency syndrome (letter). J. Amer. med. Ass. 256 (1986) 3092-3098 8. Hance, A. l-. V. Grandchamp, B. and Leuy-Frebault, D. Lecossier, j. Rauzier, and B. Gicquel: Detection and identification of mycobacteria by amplification of mycobacterial DNA. Molec. Microbiol. 3 (1989) 843-849 9. Styblo, K.: Overview and epidemiological assessment of the current global tuberculosis situation with an emphasis on control in developing countries. Rev. Infect. Dis. 11, Suppl, 2 (1989) 5339-5146 10. Quinn, C. K.: Interactions of the human immunodeficiency virus and tuberculosis and the implications for BeG vaccination. Rev. Infect. Dis. 11, Suppl, 2 (1989) 5379-5384
12 8
R. Ratei, W. Zaki , J. Wagner, and H. Hahn
11. Snider ir., D. E.: Research toward s global control and prevention of tuberculosis with an emphasis on vaccine development: Introdu ction. Rev. Infect. Dis. 11, Supp\. 2 (1989)
5336-5338 12. Statistisches Bundesarnt: Statistisches Jah rbuch fur die Bundesrepublik Deut schland. Metzl er-Poeschel Verlag, Stu ttgart (1978-1 988) Dr. Richard Ratei, Institur fur Med . Mi krob iologie und Infektionsimmunologie, Hindenburgda mm 27, 1000 Berlin 45, Germany
Diagnosis, Prevalence and Drug Resistance of Mycobacteria in HIV-Positive and HIV-Negative Patients of an Urban Population in Germany RICHARD RATEI, WALEED ZAKI, JUTTA WAGNER, and HELMUT HAHN Institut fiir Medizinische Mikrobiologie und Infektionsimmunologie, Freie Universitat Berlin, 1000 Berlin, Germany
With 1 Figure· Received August 2, 1991 . Revision received November 22, 1991 . Accepted Janu ary 21, 1992
Summary In a survey of 3004 clinical specimens from 1112 HIV-negative and 679 clinical specimens from 140 HIV-positive patients which were submitted to our mycobacter iallaboratory, we have analysed the different diagno stic approaches concerning mycobacterial disease in these two populations. We have assessed specimen-rype, culture result, microscopical diagnosis and prevalence in relation to HIV status. Isolation rates of mycobacteria were highest in blood culrures from HIV-positive patients . 10 out of 140 HIV-positive patients had positive mycobacterial culture results. All 10 had positive blood cultures, but only three of them were positive for M. tub erculosis. Zusammenfassung Anhand von 3004 mikrobiol ogischen Proben von 1112 HIV-negat iven Parienten und von 679 mikrobiologischen Proben von 140 HIV-positiven Patienten wurden die unterschiedlichen diagnostischen Vorgehensweisen beztiglich einer Mykobakterieninfektion bei diesen beiden Populationen unrersucht. Hierb ei wurden sowohl die Art des eingesandten Materials, das Kulturergebnis, die mikro skopische Diagnose, als auch die Pravalenz berticksichtigt und in Bezug zum HIV-Statu s gesetzt. Die hochste kulturelle Nachweisrate fand sich in Blutkulturen von Hl'V-Paticnren. Von insgesamt 140 HIV-positiven Patienten konnte bei 10 Patienten ein kultureller Nachwei s gefuhrt werden. Bei jedem dieser 10 Patienten konnten Mykobakterien in der Blutkultur nachgewiesen werden. Nur drei dieser Isolate waren M. tub erculosis. Introduction Tuberculosis has remained to be a major public health problem with an estimate of 16 million prevalent cases, one billion infected and about 8 million new cases of
R. Ratei, W.Zaki, J. Wagner, and H. Hahn
120
tub erculosis occurring annually thr oughout the world (11). The magn itude and dynamics of the world's tube rculosis problem are mainly reflected by the situation in the developing countries where the annual risk of tuberculosis infection has hardly decreased at all since 1900. In contrast, there has been a steady decline in the industrialized world down to an annua l risk of tuberculosis infection of below 0.05 % (11). The dramatic redu ction in morbidity and mortality from tub erculosis in the indu strialized countries can be attributed mainly to the socioeconomic development whi ch brought better living conditio ns, pa rticularly with regard to general health care and nutrition and the introdu ction of tu berculosta tic chemotherapy. Nonetheless, eradi cation of tub erculosis or mycobacterial diseases in the indu strialized countries is far from complete (5, 9). The total annual incidence of tub erculosis in the Federal Republ ic of Germany (FRG) declined from 48.2 11 00.000 in ] 978 to 21.9 /100.000 in 1988. In the first seven year s of th is decade, the tot al incidence dropped annually by a mean of 3.38 cases/ 100,000. During the follow ing four years, the total incidence decreased only by an annual mean of 1.96 cases/lOO,OO, leading to a flattening of the form erly steep decline of the total incidence of tuberculosis (Fig. 1). This development was almost paralleled by the incidence rates for first infections. Only the reinfection rates have been constantly decreasing with O.2-0.9/100,OOO/year (12).
60
...... nI
u
-
~
0
Gl
~
Gl
C
'u
40
0
0
~
~
.5 Cii
Total
20
First infection
Gl III
III
u
Reinfection
o 78
79
80
81
82
83
84
85
86
87
88
time (years)
Fig. 1. Incidence of tuberculosis in the Federal Republic of Germany between 1978 and 1988 (12).
Th at gives suppo rt to the notion that the eradication of tuberculosis has come to a poin t where first infections cannot be prevented with the same efficacy like they had been unt il 1984. The rate of reinfection s remained constant dur ing the same period. With the advent of HIV and AIDS, new clinical and epidemiological aspects have to be considered in the estimation of tub erculosis and mycobacterial diseases. Especially the occurrence of " mycobacteria other than tuberculosis" (M OTT ) in clinical specimens from patients with AIDS has to be evaluated in the light of the path ogenesis of mycobacteria-associated symptoms like dia rrhea and wasting (10, 7).
Mycobacteria in HIV-Positive and HIV-Negative Patients
121
In the following report we have summarized the data gathered by our mycobacterial laboratory to present an analysis on the prevalence, the drug resistance and sensitivity and the identification of mycobacterial isolates from a heterogeneity of clinical specimens from two groups of patients thought to be representative of an average size mycobacterial laboratory in the urban area of Berlin.
Material and Methods Clinical specimens. Between July 1", 1991 and December 31", 1991 3683 clinical specimens from Hl V-positive and HIV-negative patients were routinely subjected to mycobacterial diagnosis in our laboratory. All patients were either in- or outpatients from two major and various minor hospitals belonging to the western boroughs of Berlin. HIV status, origin and type of specimen were assesed according to informations given by the requesting clinician. Culture and identification. Depending on the type of specimen and the clinical situation, the materials were concentrated by centrifugation, pretreated and then submitted to microscopy and culture. Microscopy is routinely performed with either Auramin or Ziehl-Neelsen staining, or both. Three different culture media are inoculated separately: LoewensteinJensen, Gottsacker and Stonebrink. Inoculated cultures are incubated for 8-12 weeks at 37°C in a COrenriched atmosphere and checked weekly for growth. For further identification, cultural isolates are routinely evaluated by the following reactions: colony morphology, growth at 25 °C/42°C, catalase activity, niacin production, nitrate reduction, urease Tween 80 hydrolysis, presence of arylsulphatase, pyrazinamidase, pigment production, iron uptake, resistance to INH I and TCH 2 • Testing for drug resistance and sensitivity. Using a combination between the proportion method according to Canetti and the MIC-method resistance and sensitivity to the following tuberculostatic drugs were routinely tested: isoniazid (0.25-1.0 ug/ml), streptomycin (4.0-8.0 ug/ml), ethambutol (1.0-2.0 ug/rnl) , rifampicin (8.0-32.0 ug/ml), capreomycin (16.0-32.0 Itg/ml), cycloserine (16.0-32.0 ug/ml) and prothionamide (16.0-32.0 ug/ml) (4). Data processing and statistics. All data, including patient identification, type of specimen, microscopy, HIV status, culture period, species diagnosis and drug resistance were gatherd in a data bank on an EXL computer (© 1988 Prime Computer) running under the Unix operating system V Release 3.1. Data processing and statistics were performed with P-STAT 8, version 2.11 (© 1989, P-STAT, Inc.) software.
Results Specimens and culture results within patient status We screened 3683 clinical specimens from 1252 patients, 140 (11.2%) of whom were known to be HIV-positive and 1112 (88.8%), HIV-negative. With 3004 (81.6%) specimens from HIV-negative patients the specimen/patient ratio as a measure for the diagnostic surveillance scored 2.7 whereas in the HIV-positive population a mean of 4.9 specimens/patient is indicative of the different and much more vigilant diagnostic surveillance afforded to these patients (Table 1). The heterogeneity of clinical specimens from these two populations and the cultural results have been summarized in Table 2.
I 2
isoniazid (10 Itg ml- I ) thiophene-2-carboxylic acid hydrazide (1 Itg ml")
122
R. Ratei, W. Zaki,
J. Wagner, and H. Hahn
Table 1. HIV status, number of patients and specimens and specimen/patient ratio Status
Patients
Specimens
specimens! patient
HIV-negative HIV-positive
1112 (88.8%) 140 (11.2%)
3004 (81.6%) 679 (18.4%)
2.7 4.9
Table 2. Heterogeneity of clinical specimens sorted according to patients status and cultural result. The original 70 different specimen types have been summarized into 20 categories: B.secret. = Bronchialsecretion, B.lavage = Bronchiallavage, P.fluid = Pleural fluid, Gaspirate = gastric aspirate, CSF = cerebrospinal fluid, Misc. = Miscellaneous, ns. = not specified Culture negative Specimen Sputum B.secret. B.lavage P.fluid G.aspirate Ascites Blood CSF Lymphnode Urine
Total
HIV status positive negative
599 40 168 165 173 17 205 87
507 37 125 160 158 16 19
12
1341
10 1245
39 3 37 3 10 1 163 15 1 83
2 11
1 3
8
72
Culture positive HIV status negative positive 12
1
4 2 4
2 1 23
1 9
4
Biopsies
Liver Duodenum Ileum Colon Sigma Rectum
1
2
1
1
6 7 13
2 5 10
4 2 3
Stool Swabs Misc. ns.
597 97 166 15
363 85 126 15
228 5 34
1 5 6
5 2
Total
3683
2960
640
44
39
Obvious differences are seen in the distribution of specimens obtained for the diagnosis of mycobacterial disease from these two populations. They clearly reflect the underlying pathogenetic principles of tuberculosis in the HIV-negative not immunocompromised patient and mycobacterial disease due to ubiquitous mycobacteria in the HIV-positive immunocompromised patient. Thus the diagnostic mainstay in the
Mycobacteria in HIV-Positive and HIV-Negative Patients
123
HIV-positive population is carried by culturing stool and blood specimens, whereas in the HIV-negative population, specimens from the respiratory tract and urine are much more common. For the HIV-negative patients, specimens most commonly associated with positive cultural results are sputum, urine and swabs from various sites as compared to blood and stool for HIV positive patients. The exact origins of all 83 clinical specimens which have been culturally positive for mycobacteria are shown in Table 3.
Table 3. Depiction of clinical specimens and positive culture results according to HIV status. Swabs ns. = swabs not specified, wound sw. = wound swab, punctate ns. = punctate not specified, tissue ns. = tissue without any further specification, gastric a. = gastric aspirate, wound sec. = wound secretion, cystic f. = cystic fluid, biopsy ns. = biopsy not specified, bone m. = bone marrow Specimen Swab ns. Wound sw. Bronchial!. Blood Tissue ns. Bone m. Lymphnode Hastric a. Biopsy ns.
HIV status negative positive 3 4
2 2 23
1 1 1 4 2
Specimen
HIV status negative positive
Liver biopsy Punctate ns. Pleural fluid Pus Sputum Stool Wound sec. Cystic f. Urine
1 2 2 12 1 1 1 9
1
1 5 4
The overall isolation rate of 83 (2.25%) positrve cultures from 3683 specimens composed of 70 different specimen types refers to only 19 different specimen types. The overall isolation rate of 35 out of 1252 patients (2.8%) includes 25 HIV-negative patients resulting in a prevalence of 2.25% for this population and 10 HIV-positive patients with a significantly (Yates X 2 = 8.38; pr = 0.004) higher prevalence of 7.14% (Table 4).
Table 4. Prevalence of mycobacteria in HIV-positive patients is significantly higher than in HIV negative patients (Yates X 2 = 8.38; pr = 0.0004) Status
Patients
Positive culture
Prevalence
HIV-negative HIV-positive
1112 140
25 10
2.25% 7.14%
Total
1252
35
2.8%
124
R. Ratei, W. Zaki, J. Wagnet, and H. Hahn
Results of microscopy and culture and patient status 3
Of 3683 clinical specimens, 1264 (34.3 % ) were subjected to microscopical diagnosis with only 1.9 % (n = 25) positive results. Microscopy was not done in 65.7% (n = 2419 ) of all specimens due to insufficiency of specimen type or specimen quantity. From 83 culturally positive specimens in both groups of patients, only 54 had been microscoped of which 44 were false-negative, i.e., negative microscopy and positive cultural result . Onl y 10 specimens had positive microscopic results which were confirmed later on by positive culture result. Thus , sensitivity of microscop y is expressed by the ratio of trul y positive microscopies (n = 10) and the number of positive culture results (n = 54 ). .. . truly positive microscopies 10 0 18 .. = - = . Senstuutty = postttue culture results 54 Truly negative microscopies were attested by negative culture results in 777 specimens of HIV-negative patients and in 418 specimens of HIV-= positive patients. In 15 specimens, positive microscop y was not confirmed by the culture result. Accordingly, specificity of microscopy is exp ressed by the ratio of truly negative microscopies (n = 1195 ) and the number of negative culture results (n = 1210 ). S eciticit = truly ne?ative microscopies = 1195 = 0.99 P I' Y negatt ue culture results 1210 The accurac y of microscop y in detecting mycobacteria is expressed by the number of truly positive microscopies divided by the number of all positive microscopies including tho se which were not been confirmed later on by a positive culture result. I
_
A ccurac) -
trul y positiv e microscopies II .. . . a postttue mt croscopies
10 = -25 = 0040
Species identification and pati ent status"
In the group of HIV-negative patients, we cultured M. avium from specimens of four pati ents, M. bovis and M. gordonae in one patient each and M. tuberculosis in 19 patients, which amounted to a total of 25 isolates in this group. Only two species were isolated in the HIV-positive group of wh ich 70% (n = 7) were M. avium and 30 % (n = 3) were M. tuberculosis. Drug resistance and sensitivity:
The resistance of the 11 M. avium strains was in accordance with the well known multiple resistance of atypical mycobacteria to primary antituberculosis drugs like INH 6 , streptomycin, ethambutol and rifampicin. All M. avium strains showed susceptiblity to cycloserine at a concentration of between 16.0 and 32.0 ug/ml. M. tuberculosis strains showed consistent susceptibility to all the tested primary antituber-
see Table 5 see Table 6 5 see Table 7 (, Isoniazid 3
4
Mycobacteria in HlV-Positive and HlV-Negative Patients
125
Table 5. Patient status within culture result. Cell contents are; Total N = cell counts, Row Pet = row percent, Col Pet = column percent, and Tot Pet = percent of total N Culture negative
Culture positive
Microscopy
Total
H1V status negative positive
HIV status negative positive
no Total N Row Pet Col Pct Tot Pct
2419 100.0 65.7 65.7
2176 90.0 73.5 59.1
214 8.8 33.4 5.8
22 0.9 50.0 0.6
7 0.3 17.9 0.2
negative Total N Row Pct Col Pet Tot Pct
1239 100.0 33.6 33.6
777 62.7 26.2 21.1
418 33.7 65.3 11.3
18 1.4 40.9 0.5
26 2.1 66.7 0.7
positive Total Row Pet Col Pet Tot Pct
25 100.0 0.7 0.7
7 28.0 0.2 0.2
8 32.0 1.2 0.2
4 16.0 9.1 0.1
6 24.0 15.4 0.2
Total N Row Pct Col Pet Tot Pet
3683 100.0 100.0 100.0
2960 80.4 100.0 80.4
640 17.4 100.0 17.4
44 1.2 100.0 1.2
39 1.0 100.0 1.0
Table 6. Species identification and patient status of the 35 patient isolates on the basis of 83 culturally positive specimens Patient status H1V negative H1V positive Total
M. avium
4 7 11
M. bovis
M. gordonae
M. tuber.
Total
1
1
19 3
25 10
21
35
culosis drugs as well as to cycloserine and PTH 7 • Resistance to streptomycin at a concentration of 4 to 8 ug/ml was found in two strains. Two other strains only showed sensitivity to streptomycin at a concentration of 8 ug/ml and not at 4 ug/rnl. These two strains were considered to be in the borderline of resistance.
7
Prothionamide
126
R. Ratei, W.Zaki, J.Wagner, and H. Hahn
Table 7. Drug resistance and sensitivity of 35 isolates of mycobacteria. INH = isoniazid, PAS = p-aminosalicylic acid, PTH = prothionamide Antibiotic
M. autum
M. bovis
M. gordonae
M. tuber.
Total
INH
resistant sensitive
11 22
12 23
Streptomycin
borderline resistant sensitive
10 1
1
2 2 18
20
22
11 24
22
11 24
5 22
11 24
22
1 34
22
35
2 13
Ethambutol
resistant sensitive
11
Rifampicin
resistant sensitive
11
1
Capreomycin
resistant sensitive
11
Cycloserine
resistant sensitive
1 11
PTH
sensitive
11
1
Discussion The diagnosis of tuberculosis does not only rely upon the isolation and identification of acid-fast rods in clinical specimens alone but is mainly based on clinical grounds. In fact, tuberculosis of the respiratory system in the Federal Republic of Germany in 1986 and 1987 was diagnosed in 50% of cases (10.71100,000) on clinical grounds alone, without the isolation of tuberculosis bacteria (12). Nonetheless, it has been shown recently that for the eradication of tuberculosis it is of paramount importance to isolate and identify the mycobacterial strain in each case in order to track down and control infection routes (6). With the advent of HIV infections, the occurrence of atypical mycobacteria in clinical specimens must be borne in mind and their pathogenetic role in the immunocompromised host has to be meticulously evaluated (3). As one can see from the specimen-patient ratio (Tabl 1), the surveillance and diagnostic coverage by taking specimens to be examined for mycobacteria is much better
Mycobacteria in Hlv-Positive and HIV-Negative Patients
127
for the HIV-positive population than it is for the HIV-negative population. The assessment of prevalence rates of positive mycobacterial culture results in these two populations (Table 4) must therefore be considered with reservation because of different diagnostic approaches. Thus, the question arises whether the rate of tuberculosis cases with positive isolation of M. tuberculosis in the HIV-negative population would increase with a more stringent diagnostic approach including more and better specimens and enhanced detection methods, e.g. the polymerase chain reaction (8, 1). In the HIV-positive group, the isolation rate of mycobacteria from blood cultures was particularly high: of 140 patients in whom multiple and various cultures were performed, three patients had positive blood cultures for M. tuberculosis and seven, for M. avium. This accounts for an overall isolation rate of mycobacteria in blood cultures of 7.14%, of 0.21 % for M. tuberculosis and of 0.5% for M. avium. This is in contrast to the isolation rates mentioned by Bonza et al. who have described a total of 67 cases of tuberculosis among 100 patients with AIDS and an isolation rate of M. tuberculosis from blood cultures of 64% in these patients (2). In our survey, M. tuberculosis was cultured in only three HIV -positive patients out of 140 in whom blood cultures had been performed. The low isolation rate of M. tuberculosis in HIV-positive patients in this survey as compared to Bonza et al. can most certainly be attributed to a lower incidence of tuberculosis in Germany and to a different composition of the HIVpositive population with regard to socioeconomic status, homosexuality and drug abuse.
References 1. Boeddingbaus, T. Rogall, T. Flohr, H. Bloecker, and E. C. Boettger: Detection and identification of mycobacteria by amplification of rRNA. J. Clin. Microbiol. 28 (1989) 1751-1759 2. Bonza, E., C. Martin-Scapa, j. C. L. Bernaldo de Quiros et al.: High prevalence of tuberculosis in aids patients in spain. Europ. J. Clin. Microbiol. Infect. Dis. 7 (1988) 785-788 3. Braun, M. M., R. H. Byers, W. L. Heyward et al.: Acquired immunodeficiency syndrom and extrapulmonary tuberculosis in the united states. Arch. Intern. Med. 150 (1990) 1913-1916 4. Canetti, G.: Mycobacteria: Laboratory methods for testing drug sensitivity and resistance. Bull. Wid Hlth Org. 29 (1963) 5565-578 5. Gracey, D. R.: Tuberculosis in the world today. Mayo Clin. Proc. 63 (1988) 1251-1255 6. Gross, T. P., P. R. Silverman, A. B. Bloch et al.: An outbreak of tuberculosis in rural Delaware. Amer. J. Epidem. 129 (1989) 362-371 7. Guarner, j., C. del Rio, and B. Slade: Tuberculosis as a manifestation of the acquired immunodefiency syndrome (letter). J. Amer. med. Ass. 256 (1986) 3092-3098 8. Hance, A. l-. V. Grandchamp, B. and Leuy-Frebault, D. Lecossier, j. Rauzier, and B. Gicquel: Detection and identification of mycobacteria by amplification of mycobacterial DNA. Molec. Microbiol. 3 (1989) 843-849 9. Styblo, K.: Overview and epidemiological assessment of the current global tuberculosis situation with an emphasis on control in developing countries. Rev. Infect. Dis. 11, Suppl, 2 (1989) 5339-5146 10. Quinn, C. K.: Interactions of the human immunodeficiency virus and tuberculosis and the implications for BeG vaccination. Rev. Infect. Dis. 11, Suppl, 2 (1989) 5379-5384
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R. Ratei, W. Zaki , J. Wagner, and H. Hahn
11. Snider ir., D. E.: Research toward s global control and prevention of tuberculosis with an emphasis on vaccine development: Introdu ction. Rev. Infect. Dis. 11, Supp\. 2 (1989)
5336-5338 12. Statistisches Bundesarnt: Statistisches Jah rbuch fur die Bundesrepublik Deut schland. Metzl er-Poeschel Verlag, Stu ttgart (1978-1 988) Dr. Richard Ratei, Institur fur Med . Mi krob iologie und Infektionsimmunologie, Hindenburgda mm 27, 1000 Berlin 45, Germany