EP0532584A1 - Dna for diagnosing pneumocystis carinii - Google Patents

Abstract

A method of analyzing a DNA sample taken from a patient's respiratory secretions for the detection of Pneumocystis carinii involves amplifying the polynucleotide sequence derived from P. carinii by a polymerase chain reaction and detecting the amplified sequence if present. Two DNA sequences are provided, as well as a number of pairs of oligonucleotide primers, including in particular the pair (I).

Description

DNA FOR DIAGNOSING PNEUMOCYSTIS CARINII Introduction

Pneumocystis carinii is established as the prime cause of opportunistic pneumonia in patients with AIDS and those immunosuppressed on oncology and transplant units. Debate over the taxonomy of

P. carinii continues and the fastidious nature of the parasite still demands the use of microscopy after histochemical staining or immunofluorescence for the detection of the parasite in diverse forms of lung samplings [1,2]. The requirement for observer

expertise is significant and limits the diagnostic power of these techniques. There are sound theoretical grounds for believing that DNA amplification using the polyneraβe chain reaction (PCR) [3] might provide both a specific and sensitive means of identifying the parasite in clinical samplings and one that finally might be amenable to automation. Other applications of DNA amplification to the study of the epidemiology of P. carinii infection are also evident.

European Patent Specification 327390

describes DNA sequences produced by recombinant DNA technology from an experimental rat model which

hybridise to DNA of P.carinii but not to mammalian DNA. These DNA sequences were present as inserts in plasmids of which two were designated pAZ 102 and pAZ 112. This invention results from further work described herein:- A. We have sequenced the inserts of pAZ 102 and pAZ 112. The sequences are set out in Figures 1 and 3. B. Using suitable oligonucleotide primers we have amplified, by the polymerase chain reaction (PCR) technique, P. carinii DNA from infected rat lung

samplings, to an extent that the amplified DNA was easily detectable by staining an electrophoresis gel. In the same way, we have amplified P. carinii DNA from infected human lung samplings.

C. Analysis of the amplified DNA from human samples has shown significant sequence differences from the infected rat material. The human sequence is set out in Figure 1, and the similarities and differences between human and rat sequences are highlighted.

D. The human sequence, leads to improved

oligonucleotide primers which more efficiently amplify P. carinii DNA of human origin.

E. We have developed a more sensitive detection system, involving hybridising the amplified DNA to a labelled probe which probe is part of the sequence determined in C. intermediate the two primers. By these means we are able to detect P.carinii DNA, and thus to diagnose infection by P.carinii. in patients who do not (yet) show clinical symptoms.

In one aspect, this invention provides, as new chemical compounds, the nucleic acid sequences shown in Figures 1 and 3, single and double chain fragments thereof at least 15 nucleotides in length, and nucleic acid sequences and fragments having at least 90% homology thereto. These result from steps A. and C. above.

In another aspect, the invention provides a method of assaying a sample of DNA from respiratory secretion of a patient possibly infected with

Pneumocystis carinii, which method comprises using a pair of oligonucleotide primers based on the sequences shown in Figure 1 or Figure 3 to amplify by a

polymerase chain reaction a polynucleotide sequence derived from P. carinii if such sequence is present in the sample, and detecting the amplified sequence if present. This is step B. above, and steps D. and E. constitute preferred features of the method. For maximum efficiency and specificity of the

PCR reaction, the choice of oligonucleotide primers is critical. The primers must be based on the sequence to be amplified and may be identical to the two ends.

However, identity is by no means essential (R. Sommer and D. Tautz, Nucleic Acids Research, Vol. 17, No. 16,

1989, p 6749). Generally the two or three nucleotides at the 3'-end of the primer, and at least 50%

(preferably at least 90%) of all the nucleotides of the primer, are homologous to the sequence to be amplified.

The primers are partly or completely homologous to particular sites of the sequence to be amplified. For maximum efficiency of the PCR reaction, the location of those sites is also important.

Although most primers will work with varying degrees of success, general guidelines for obtaining useful primers are found in the literature (see Saiki R. K. et al., The Polymerase Chain reaction in Genome Analysis

(Ed K. E. Davies) IRL, Oxford, 1988). However, the design of effective primers tends to be empirical.

Described below are one pair of primers derived from pAZ 102 that have proved outstanding; and several pairs of primers derived from pAZ 112 that have proved effective.

In other respects, the PCR conditions may be conventional. The primers may be at least 8,

conveniently about 20, nucleotides in length. The number of cycles required to achieve sufficient

amplification may be from 15 to 50. If required to improve specificity, two different pairs of primers may be used. The resulting amplified sequence has a predetermined length, and moves a predetermined

distance on an electrophoresis gel. The resulting band can be visualised, either by conventional staining techniques, or by hybridisation to a labelled probe which probe is homologous to part or all of the known sequence being amplified.

Reference is directed to the accompanying Figures, in which:-

Figtire 1 comprises sequence data on different DNA samples. Row 1 entitled "Rat" is from lung samplings from a rat infected with P. carinii.

Row 2 entitled "Human" is from lung samplings of infected humans. Secondary structure has been taken into consiiderationand gaps (-) introduced to obtain maximum alignment. Numerous differences between human and rat sequences are shown boxed.

Figure 2 is a diagram of the circular plasmid pAZ 112 showing certain features including the

positions of polynucleotide primers used in PCR.

Figure 3 comprises the complete sequence of the insert of pAZ 112, with the oligonucleotide primers marked. R/C means reversed and complemented, i.e. the actual sequence of the primer is the reverse and complementary to that marked.

Table 2 lists the oligonucleotide primers referred to in Figures 2 and 3.

Table 3 lists the primer combinations successfully used by us and the approximate size of the resulting amplification product.

The following Examples illustrate the invention. Example 1 relates to DNA from the plasmid pAZ 102 whose sequence is shown in Figure 1. Example 2 relates to DNA from the plasmid pAZ 112 whose sequence is shown in Figure 3. Example 3 reports a clinical trial following the method of Example 1.

Example 1 Methods Cloning and sequencing of part of the gene coding for the large sub-unit of the mitochondrial ribosomal RNA from P. carinii

P. carinii pneumonia was induced in the tat model and DNA extracted and cloned from a parasite enriched fraction as previously described [4].

P.carinii specific sequences were confirmed by characteristic in situ hybridisation patterns and recombinant plasmid pAZ102 was selected as a candidate mitochondrial sequence because of strong signals derived in dot blot hybridisation studies on infected saπples. The recombinant plasmid pAZ102 (insert 570 bp) was sequenced using Sanger's chain termination method and the Sequenase kit (United States Biochemical Corporation, Cleveland, USA), ³⁵S(Amersham, UK), Sequagel (National Diagnostics,

Manville, USA). The ENA sequence was compared with those available in several databases including EMBL and Genbank. From the sequence data on pAZ102 and comparative analysis of the databases, the fragment was identified as a portion of the gene coding for the large sub-unit of the ndtochoridrial, ribosomal RNA of P.carinii and this showed significant homology with fungal sequences (manuscript in preparation).

Oligonucleotide primers

Two sequences of moderate conservation that were specific to P.carinii were selected for construction of oligonucleotide primers for the

polymerase chain reaction:- pAZ102 - E:-5'-GATGGCIGTTTCCAAGCCCA-3';

pAZ102-H:-5'-GTGTACGTTGCAAAGTACTC-3'. An oligonucleotide for confirmatory Southern hybridisation on amplification products was chosen, pAZ102-L1. Subsequently a new internal oligonucleotide specific to human P.carinii sequences was constructed, pAZ102-L2 (Table 1) .

Template DN A

i) Samplings for ENA anplification using our oligonucleotide primers

comprised a) pulmonary lavage sanplings from 3 humans and 3 rats with P.carinii pneumonia documented by methenamine silver staining and microscopy, and b) isolates from a series of organisms including some potential pulmonary pathogens: Candida (an albicans and a non-albicans strain) , Cryptococcus neoformans, Mycobacterium tuberculosis.

Saccharomyces cerevisiae and Aspergillus nidulans.

ii) Template DNA was prepared from each sa mple by proteinase K digestion in the presence of SDS and EDEA followed by phenol/chloroform/ether extraction, and ethanol precipitation.

LNA amplification

Using primers pAZ102-E and pAZ102-H the template samples, together with control samples without template underwent 40 cycles of amplification performed with denaturation at 94°C for 90 seconds, annealing at 50°C for 90 seconds and extension at 72°C for two minutes (Techne, UK) . The ENA a mplifica tion reaction mixture (50 μl) contained 50m M KCl, 10 mM Tris, pH

8, 0.01% (w/v) gelatin, 3 mM MgCl₂, 400 μM dNTPs (Boehringer Mannheim, UK) , 0.4-1.0 μM oligonucleotide primer and 3 units of Amplitaq (Perkin Elmer Cetus, UK) .

To avoid the possibility of false negative results in the human clinical saπples, i.e. failure to detect the specific amplification product for technical reasons, we carried out a parallel polymerase chain reaction on each sample using primers derived from the human anti-thrombin gene, exon 2

These primers, (AT1: -5'-

GTTGCAGCCTAGCTTAACTTGGCA-3 ' ; AT4 : -5 ' -GGTTGAGGAATCATTGGACTTG-3 ' ) allowed amplification to take place using human genomic DNA as template. In each of the clinical samples the 500 bp specific product was detected,

demonstrating efficient amplification.

The potential problem of contamination in PCR was monitored by

systematic use of the following techniques: i) including several negative control sanples with no added tenplate ENA; ii) by the use of UV- irradiation of the PCR reaction mixes prior to the addition of the template

ENA [5] , iii) the use of separate disposable inicrocapillaries for the addition of each template (Laser Laboratory Systems Ltd, UK) . The control samples remained negative in all experiments.

Amplified products (10 μl) were electrophoresed in 1.5% agarose gels and visualised after ethidium bromide staining by ultraviolet light. The gel was Southern blotted on to Hybond N (Amersham, UK) and hybridised with ³²P end-labelled internal primer at 46°C (pAZ102-L1) or 40°C (pAZ102-L2) for 3 hours[6] . Filters were subsequently washed at high stringency at 54°C (PAZ102-L1) or 48°C (pAZ102-L2) and filters exposed to radiographic film at -80°C with intensifying screens. The expected anplification product was 355bp long in the rat derived parasite, that from the human derived parasite being 9 bp shorter.

Sequencing of products of DNA amplification

The PCR product was gel purified and recovered from the agarose gel using Geneclean (Bio 101, Inc) . The purified ENA was heat denatured and seqυenced as described above using primers pAZ102-H or pAZ102-E at

20praole/μl.

Results

The oligonucleotide primers derived from rat P.carinii produced efficient anplification of specific sequence from both rat and human hosts, shown by ethidium brαidde staining but none from the range of other organisms including some potential pulmonary pathogens.

The internal oligonucleotide, pAZ102-L1, derived from the rat P.carinii. produced strong hybridisation signals on Southern hybridisation with amplified products from the infected rat lungs, but weak signal, at high stringency, with the amplified product derived from human samples although these were visible on ethidium bromide staining. Direct sequencing of the amplified products from each of the rat and human samples allowed comparison of their sequence and demonstrated limited but

consistent differences between the P.carinii DNA from these two hosts which included 5 base changes in the sequence of the internal oligonucleotide pAZ102-L1 (Table 1) . An oligonucleotide specific to the human derived organisms was constructed, pAZ102-L 2, which showed strong hybridisation with the amplified product from human P.carinii and conversely showed weak hybridisation with the rat P. carinii amplified product. It produced no hybridisation with the PCR products of the range of other organisms tested.

Studies using serial dilutions of human derived P. carinii template DNA indicated that the application of oligoblotting with pAZ102-L2 to amplified DNA products increased the sensitivity of detection by at least 100 fold over visualisation by ethidium bromide staining.

The P.carinii oligonucleotide primers successfully amplified specific PCR product from bronchoscopic alveolar lavage samplings from 10 HIV-positive individuals with pneumocystis pneumonia as documented by positive methenamine silver staining on the lavage samples. Lavage samples from 5 immunocompetent subjects were studied as controls. These failed to show specific PCR product by ethidium bromide staining or oligoblotting.

Discussion

We have characterised a portion of the gene coding for the large sub-unit of mitochondrial, ribosomal RNA from P.carinii and comparative analysis of this indicates significant homology with fungal sequences . This result accords with data we have on other mitochondrial genes

and the observations of other groups on ribosomal RNA[7]. We have identified P.carinii specific sequences from which we have constructed oligonucleotide primers which allow efficient amplification of part of this ribosomal RNA gene from P. carinii infecting both rat and human hosts but not from a range of other organisms including some potential pulmonary pathogens. These results indicate the specificity of the amplif ied products to P. carinii , confirmed on Southern hybridisation with internal oligonucleotide pAZ102-L1 derived from the rat sequence and applied to the rat pulmonary samplings . Results from the human samplings suggested the likelihood of differences in sequence between the amplified products from the rat and human . This was confirmed by comparison of sequences which indicated limited, but consistent differences.

This finding is highly relevant to the hopes for developing DNA amplification as a diagnostic tool in clinical medicine. We have shown that our oligonucleotide primers can be used to identify the presence of P.carinii in a number of bronchoalveolar lavage samples. By using our second internal oligonucleotide . pAZ102-L2 , which is specific to human P.carinii. the sensitivity of detection of amplified product is considerably increased. This method shows great potential for use on non-invasive samples such as induced sputum where parasite numbers are lower. It will not only be valuable in diagnosis but also in addressing questions relevant to the epidemiology of P.carinii .

The application of DNA amplification to diagnosis will require careful calibration to ensure that levels of P. carinii in keeping with clinical pneumonia can be distinguished from lesser degrees of colonisation that are likely to occur in the immunodeficient before clinical disease is manifest. Such methods of calibration of DNA amplification are becoming available [8,9] and their application to diagnostic studies on P.carinii in diverse clinical samplings , including lavage and induced sputum, are now required . Example 2

For pAZ 112 we used the techniques described in Example 1, and the oligonucleotide primers given in Table 2 in the combination set out in Table 3. We achieved amplification by PCR of the sequences of pAZ 112 shown in Figure 3.

Example 3

Clinical Specimens

Alveolar lavage samples were obtained from 47 patients investigated by bronchoscopy at the Churchill Hospital, Oxford, and at the Middlesex Hospital,

London.

Thirty seven patients were immunosuppressed, either by HIV infection (33) or by treatment for lyraphoma (2), vasculitis (1) or leukaemia (1). All patients had symptoms of acute respiratory illness with one or more of the following features: abnormal chest signs, arterial hypoxaeraia, or abnormal chest

radiograph. The 10 remaining patients were

immunocompetent, undergoing bronchoscopy to investigate various respiratory disorders. Routine microbiological and cytological analysis including methenamine silver staining was performed on each lavage and an aliquot reserved for the DNA amplification study, performed as described in Example 1.

Results

On the basis of clinical progress, response to treatment with nebulised pentamidine (20 cases) or cotrimoxazole (7 cases) and results of standard investigation including methenamine silver staining, the 47 patients were eventually categorised into four groups (Table 4); 16 immunosuppressed patients with a positive diagnosis of pneumocystis pneumonia by silver staining on lavage and response to treatment, 6 immunosuppressed patients with clinical response to treatment, but negative silver stains on lavage, 15 immunosuppressed patients with neither response to treatment nor positive silver staining on lavage and in whom an alternative diagnosis to account for the respiratory disease was available in 12, and the 10 immunocompetent patients from the routine bronchoscopy list. The results of DNA amplification assayed by the visualisation of a 346 base pair DNA band after a) ethidium bromide staining and b) autoradiography after oligoblotting were compared with these clinical, data categorisations (Table 4) .

No p.carinii DNA was detectable in the samples from the immunocompetent group. All of the 16 immunosuppressed individuals with P.carinii identified by methenamine silver staining on alveolar lavage had amplified P.carinii DNA visible by both ethidium bromide staining and oligoblotting. Of the 6

individuals judged to have had pneumocystis pneumonia by clinical symptoms and response to treatment but not confirmed by identification of parasites in methenamine silver stained lavage samples, 4 were positive using DNA amplification - both by ethidium bromide staining and oligoblotting - and 2 negative by both methods.

Lesser degree of P. carinii infection were detected in the oligoblots - but not by ethidium bromide staining - of the DNA amplification product in lavage samples from 3/15 of the immunosuppressed subjects without pneumocystis pneumonia. The intensity of the signal was less than that obtained from patients with acute P.carinii infection but significantly greater than a barely visible signal obtained in 4 other samples from this patient group, and from 3 of 12 preliminary washings of the bronchoscope after routine cleaning and sterilisation.

Example 4

The method of Example 1 was extended to test induced sputum samples. Fifty one episodes of acute respiratory illness in immunosuppressed HIV-infected individuals were studied. Bronchoscopic alveolar lavage was obtained from each patient and in thirty seven instances induced sputum was also obtained.

Samples were examined by routine microbiological and cytological methods, including methenamine silver staining for P.carinii; a part of each sample was reserved for DNA amplification. DNA was extracted from 1 ml lavage or sputum by proteinase K digestion (1 rag/ml final concentration of proteinase K, in the presence of 10 mmol EDTA, pH 8.0 and 1% weight/volume sodiumdodecylsulphate, at 50°C for 16 hours and

phenol/chloroform extraction. DNA amplification was done with the oligonucleotide primers pAZ102-E and pAZ102-H, with denaturation at 94°C for 90 s, annealing at 55°C for 90 s, and extension at 72°C for 2 min (40 cycles). The amplification products were subjected to electrophoresis in 1.5% agarose gel and the specific P.carinii sequence (346 base pairs) was identified by visualisation with ultraviolet light after ethidium bromide staining or by oligohybridisation, after

Southern transfer and autoradiography with the internal primer pAZ102-L2. Scoring of the DNA bands was done without knowledge of the results of silver staining or of final clinical diagnosis, which was assessed by clinical features and response to treatment with cotrimoxazole or pentamidine.

The results from the paired lavage and sputum samples are summarised in the table 5 below. All 14 patients who had a final clinical diagnosis of

pneumocystis pneumonia and who also had a positive silver stain on lavage, had a strong signal of

amplified pneumocystis DNA from both the lavage sample and sputum. In 5 other patients with a final clinical diagnosis of pneumocystis pneumonia but negative silver stains, 4 were strongly positive by DNA amplification in alveolar lavage; 3 of these 4 were also positive in induced sputum. Silver stain was positive in only one third of sputum samples from cases of pneumocystis pneumonia.

Positive signals of amplified DNA can be categorised as strong (visible after ethidium bromide staining of the agarose gel) or weak (visible only on autoradiography after oligoblotting). Independent calibration experiments have shown that a strong signal points to 100 organisms or more in a sample, whereas a weak signal indicates from 1-2 organisms up to 100 organisms per sample. In broad terms, it may be said that patients providing samples with strong signals show clinical symptoms of pneumocystis pneumonia, whereas patients providing samples with weak signals are in the pre-cliniσal stage. Thus, in 20 cases judged to have clinical pneumocystis pneumonia, a strong DNA amplification signal was obtained in 19 (95%) of the lavage samples and in 18 (90%) of the paired sputum samples. By contrast, microscopy after silver staining could only diagnose 35% of these cases on induced sputum. The sensitivity of the DNA method is therefore excellent; it is unlikely that the single case, negative by both DNA amplification and silver staining on lavage, had pneumocystis pneumonia.

The specificity of DNA amplification may be judged from the results of another study involving 44 patients, in whom the final clinical diagnosis was of another respiratory illness (i.e. not pneumocystis pneumonia). A strong amplification signal was obtained both in the lavage and sputum samples in only one of these 44 patients; this patient had had a previous episode of pneumocystis pneumonia and returned with a further documented episode within ten weeks of the current study.

REFERENCES

1. Savoia D, Carmello P. The microscopic identification of Pneumocystis carinii. J Protozool 1989; 36: 72S-74S.

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Ognibene FP, Shelhamer J, Parillo JE, Gill VJ. Diagnosis of

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Enzymatic anplification of β-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anaemia. Science 1985; 230: 1350-1354.

4. Wakefield AE, Hopkin JM, Burns J, Hipkiss JB, Stewart TJ, Moxon ER.

Cloning of ENA frαn Pneumocystis carinii. J Infect Dis 1988; 158: 859- 861.

5. Sarkar G, Sommer SS. Shedding light on PCR contamination. Nature 1990;

343: 27.

6. Miyada OG, Wallace RB. Oligonucleotide hybridization techniques.

Methods in Enzymology 1987; 154: 94-107.

7. Edman JC, Kovacs JA, Masur H, Santi DV, Elwood HJ, Sogin ML. Ribosomal RNA sequence shows Pneumocystis carinii to be a member of the Fungi. Nature 1988; 334: 519-522.

8. Wang AM, Doyle MV, Mark DF. Quantitation of mRNA by the polymerase

chain reaction. Proc Natl Acad Sci USA 1989; 86: 9717-9721.

9. Sambrook J, Fritsch EF, Maniatis T. Quantitation of initial

concentration of target sequences. In: Molecular Cloning (2nd ed), 1989 pp 14.30-14.35, Cold Spring Harbour Laboratory Press, USA.

Claims

1. The nucleic acid sequences shown in Figure 1, single and double chain fragments thereof at least 15 nucleotides in length, and nucleic acid sequences and fragments having at least 90% homology thereto.

2. The nucleic acid sequence shown in Figure 3, single and double chain fragments thereof at least 15 nucleotides in length, and nucleic acid sequences and fragments having at least 90% homology thereto.

3. Peptide sequences transcribed from the nucleic. acid, sequences claimed in Claim 1 or Claim 2.

4. A method of assaying a sample of DNA from respiratory secretion of a patient possibly infected with Pneumocystis carinii. which method comprises using a pair of oligonucleotide primers based on the

sequences shown in Figure 1 or Figure 3 to amplify by a polymerase chain reaction a polynucleotide sequence derived from P.carinii if such sequence is present in the sample, and detecting the amplified sequence if present.

5. A method as claimed in Claim 4, wherein the amplified sequence is detected by electrophoresis and staining.

6. A method as claimed in Claim 4, wherein the amplified sequence is detected by hybridisation to a labelled, probe which probe is a nucleic acid sequence according to Claim 1 or Claim 2.

7. A method as claimed in Claim 7, wherein the probe is the 20-mer.

5'- A T A A G G T A G A T A G T C G A A A G - 3'

8. A method as claimed in any one of Claims 4 to 7, wherein the oligonucleotide primers are:-

5' - G A T G G C T G T T T C C A A G C C C A - 3' 5' - G T G T A C G T T G C A A A G T A C T C - 3' .

9. A method as claimed in any one of claims 4 to 8, wherein the respiratory secretion assayed is induced sputum.